PRIME EDITOR VARIANTS, CONSTRUCTS, AND METHODS FOR ENHANCING PRIME EDITING EFFICIENCY AND PRECISION

VARIANTS D'EDITEUR PRIMAIRE, CONSTRUCTIONS ET PROCEDES POUR AMELIORER L'EFFICACITE ET LA PRECISION D'UNE EDITION PRIMAIRE

English Abstract

The present disclosure provides compositions and methods for prime editing with improved editing efficiency and/or reduced indel formation by inhibiting the DNA mismatch repair path way while conducting prime editing of a target site. Accordingly, the present disclosure provides a method for editing a nucleic acid molecule by prime editing that involves contacting a nucleic acid molecule with a prime editor, a pegRNA, and an inhibitor of the DNA mismatch repair pathway, thereby installing one or more modifications to the nucleic acid molecule at a target site with increased editing efficiency and/or lower indel formation. The present disclosure further provides polynucleotides for editing a DNA target site by prime editing comprising a nucleic acid sequence encoding a napDNAbp, a polymerase, and an inhibitor of the DNA mismatch repair pathway, wherein the napDNAbp and polymerase is capable in the presence of a pegRNA of installing one or more modifications in the DNA target site with increased editing efficiency and/or lower indel formation. The disclosure further provides, vectors, cells, and kits comprising the compositions and polynucleotides of the disclosure. The present disclosure also provides compositions and methods for prime editing with improved editing efficiency and/or reduced indel formation with modified prime editor fusion proteins. The disclosure further provides, vectors, cells, and kits comprising the compositions and polynucleotides of the disclosure.

French Abstract

La présente invention concerne des compositions et des procédés d'édition primaire avec une efficacité d'édition améliorée et/ou une moindre formation d'indel par inhibition de la voie de réparation de mésappariement d'ADN tout en réalisant une édition primaire d'un site cible. En conséquence, la présente invention concerne un procédé d'édition d'une molécule d'acide nucléique par édition primaire qui implique la mise en contact d'une molécule d'acide nucléique avec un éditeur primaire, un ARNpeg, et un inhibiteur de la voie de réparation de mésappariement d'ADN, ce qui permet d'installer une ou plusieurs modifications sur la molécule d'acide nucléique au niveau d'un site cible avec une efficacité d'édition accrue et/ou une moindre formation d'indel. La présente invention concerne en outre des polynucléotides pour l'édition d'un site cible d'ADN par édition primaire comprenant une séquence d'acide nucléique codant un napDNAbp, une polymérase, et un inhibiteur de la voie de réparation de mésappariement d'ADN, le napDNAbp et la polymérase étant capables en présence d'un ARNpeg d'installer une ou plusieurs modifications dans le site cible d'ADN avec une efficacité d'édition accrue et/ou une moindre formation d'indel. La divulgation concerne en outre des vecteurs, des cellules et des kits comprenant les compositions et les polynucléotides de l'invention. La présente invention concerne également des compositions et des procédés d'édition primaire avec une efficacité d'édition améliorée et/ou une moindre formation d'indel avec des protéines de fusion d'éditeur primaire modifiées. La divulgation concerne en outre des vecteurs, des cellules et des kits comprenant les compositions et les polynucléotides de la divulgation.

Claims

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CLAIMS
What is claimed is:
I. A rnethod for editing a nucleic acid rnolecule by prime editing comprising:
contacting a
nucleic acid molecule with a prime editor, a pegRNA, and an inhibitor of the
DNA mismatch
repair pathway, thereby installing one or more modifications to the nucleic
acid molecule at a
target site.
2. A method for editing a double stranded target DNA sequence, comprising:
contacting the
double stranded target DNA with (i) a prime editor, (ii) a prime editing guide
RNA
(pegRNA), and (iii) an inhibitor of a DNA mismatch repair pathway,
wherein the prime editor comprises a nucleic acid programmable DNA binding
protein
(n.apDNAbp) and a DNA polymerase,
wherein the pegRNA comprises a spacer sequence, a gRNA core, and an extension
arm
comprising a DNA synthesis template and a primer binding site (PBS),
wherein the spacer sequence comprises a region of complementarity to a target
strand of
the double stranded target DNA sequence,
wherein the gRNA core associates with the nap.DNAbp,
wherein the DNA synthesis template comprises a region of complementarity to
the non-
target strand of the double-stranded target DNA. sequence and one or more
nucleotide edits
compared to the target strand double-stranded target DNA sequence;
wherein the primer binding site comprises a region of complementarity to a non-
target
strand of the double-stranded target DNA sequence,
wherein the contacting installs the one or more nucleotide edits in the double
stranded
target =DNA, thereby editing the double stranded target DNA
3. The method of claim 2, wherein the PBS comprises a region of
complementarity to a region
upstream of a nick site in the non-target strand of the target DNA sequence,
wherein the nick
site is characteristic of the napDNAbp.
4. The method of claim 1, wherein the method further cornprises contacting
the nucleic acid
molecule with a second strand nicking gRNA.
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5. The rnethod of claim 1, wherein the prime editing efficiency is
increased by at least 1.5-fold,
at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at
least 4.0-fold, at least
4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least
6.5-fold, at least 7.0-fold,
at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at
least 9.5-fold, at least
10.0 fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-
fold, at least 15-fold,
at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at
least 20-fold, at least
21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-
fold, at least 26-fold,
at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at
least 31-fold, at least
32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-
fold, at least 37-fold,
at least 38-fold, at least 39-fold, at least 40-fold, at least 41.-fold, at
least 42-fold, at least
43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-
fold, at least 48-fold,
at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at
least 53-fold, at least
54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-
fold, at least 59-fold,
at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at
least 64-fold, at least
65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-
fold, at least 70-fold, at
least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at
least 75-fold, in the
presence of the inhibitor of the DNA mismatch repair pathway compared to prime
editing
efficiency with the prime editor and the pegRNA in the absence of the
inhibitor of the DNA
mismatch repair pathway.
6. The rnethod of claim 1, wherein the frequency of indel form.ation is
decreased by at least 1.5-
fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-
fold, at least 4.0-fold, at
least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at
least 6.5-fold, at least 7.0-
fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-
fold, at least 9.5-fold, at
1 east 10 . 0-fol d, at least I 1 -fol d, at 1 east 12-fol d, at 1 east 13-fol
d, at 1 east 14-fol d, at 1 east 15-
fold, at least 16-fold, at least 17-fold, at least 18-fo1d, at least 19-fold,
at least 20-fold, at
least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least
25-fold, at least 26-
fo1d, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold,
at least 31-fold, at
least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least
36-fold, at least 37-
fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold,
at least 42-fold, at
least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least
47-fold, at least 48-
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fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold,
at least 53-fold, at
least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least
58-fold, at least 59-
fo1d, at least 60-fold, at least 61-fo1d, at least 62-fold, at least 63-fold,
at least 64-fo1d, at
least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least
69-fold, at least 70-
fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold,
or at least 75-fold, in
the presence of the inhibitor of the DNA mismatch repair pathway compared to
the frequency
of indel formation with the prime editor and the pegRNA in the absence of the
inhibitor of
the DNA mismatch repair pathway.
7. 7. The method of claim 1, wherein the purity of editing outcome is
increased by at least 1.5-
fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-
fold, at least 4.0-fold, at
least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at
least 6.5-fo1d, at least 7.0-
fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-
fold, at least 9.5-fold, at
least 10.0-fold, at least 11-fold, at least 12-fold, at least 13-fold, at
least =14-fold, at least 15-
fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold,
at least 20-fold, at
least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least
25-fold, at least 26-
fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold,
at least 31-fold, at
least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least
36-fold, at least 37-
fo1d, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold,
at least 42-fold, at
least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least
47-fold, at least 48-
fold, at least 49-fold, at least 50-fold, at least 51-fo1d, at least 52-fold,
at least 53-fold, at
least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least
58-fold, at least 59-
fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold,
at least 64-fold, at
least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least
69-fold, at least 70-
fold, at least 71-fold, at least 72-fo1d, at least 73-fold, at least 74-fold,
or at least 75-fold, in
the presence of the inhibitor of the DNA mismatch repair pathway compared to
the purity of
editing outcome with the prime editor and the pegRNA in the absence of the
inhibitor of the
DNA mismatch repair pathway, wherein the purity of editing outcome is measured
by the
ratio of intended edit/unintended indels.
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8. The rnethod of claim 1, wherein the inhibitor of the DNA mismatch repair
pathway inhibits
the expression or function of one or more proteins of the DNA misrnatch repair
pathway
(MMR proteins).
9. The method of claim 8, wherein the one or more MMR proteins is selected
from the group
consisting of MLH1, PMS2 (or MutL alpha), PMS I (or MutL beta), MLH3 (or Mud,
gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA,
RFC, EX01, POL5, and PCNA.
10. The rnethod of elairn 8, wherein the one or more MMR proteins is MLH1.
11. The rnethod of claim 10, wherein ML111 cornprises an arnino acid sequence
of SEQ ID NO:
204, or an amino acid sequence having at least 70%, at least 75%, at least
80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or up to and
including 100% sequence identity with SEQ ID NO: 204.
12. The method of claim 8, wherein the inhibitor is an antibody that inhibits
the activity of one or
more proteins of the DNA mismatch repair pathway.
13. The method of claim 8, wherein the inhibitor is a small molecule that
inhibits the activity of
the one or more proteins of the DNA mismatch repair pathway.
14. The method of claim 8, wherein the inhibitor is a small interfering RNA.
(siRNA.) or a small
non-coding rnicroRNA that inhibits the activity of the one or more proteins of
the DNA
mismatch repair pathway.
15. The method of claim 8, wherein the inhibitor is a dominant negative
variant of an MMR
protein that inhibits the activity of a wild type MMR protein.
16. The method of claim 8, wherein the inhibitor is a dominant negative
variant of MLH1 that
inhibits the activity of MLH1.
17. The method of claim 16, wherein the dorninant negative variant of MLH1
comprises one or
more amino acid substitutions, insertions, and/or deletions in an ATPase
domain compared to
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a wild type MLIT1 protein as set forth in SEQ ID NO: 204, wherein the one or
more amino
acid alterations impairs or abolishes ATPase activity of the dominant negative
variant of
MLIT1.
18. The method of claim 16, wherein the dominant negative variant of MLH1
comprises one or
more amino acid substitutions, insertions, and/or deletions in an endonuclease
domain
compared to a wild type MLH1 protein as set forth in SEQ ID NO: 204, wherein
the one or
more amino acid alterations impairs or abolishes endonuclease activity of the
dominant
negative variant of MLH1.
19. The method of claim 16, wherein the dominant negative variant of MiLH1 is
truncated at the
C terminus compared to a wild type MLI-11 protein as set forth in SEQ ID NO:
204.
20. The method of claim 16, wherein the dominant negative variant of MUT] is
truncated at the
N terminus compared to a wild type ML.H1 protein as set forth in SEQ ID NO:
204.
21. The method of any one of claims 16-20, wherein the dominant negative
variant of M:LH1
further comprises a nuclear localization signal (NLS) at the N terminus and/or
a NTS at the C
terminus.
22. The method of claim 16, wherein the dominant negative variant is (a) MLH1
E34A (SEQ
NO: 222), (b) MLH1 A756 (SEQ ID NO: 208), (c) MLH1 A754-756 (SEQ ID NO: 209),
(d)
MLITI E34A A754-756 (SEQ ID NO: 210), (e) MLI-11 1-335 (SEQ ID NO: 211), (0
MLII1
1-335 E34A (SEQ ID NO: 212), (g) MLH1 1-335 NLSs114 (SEQ ID NO: 213), (h)
MLH1
501-756 (SEQ ID NO: 215), (i) MLIT1 501-753 (SEQ ID NO: 216), (j) MLH1 461-753
(SEQ
ID NO: 218), or (k) NL,Ssv4O MLH1 501-753 (SEQ ID NO: 223), or a polypeptide
comprising an amino acid sequence having at least 70%, at least 75%, at least
80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or up
to and including 100% sequence identity with any of SEQ ID NOs: 208-213, 215,
216, 218,
222, or 223.
23. The method of claim 16, wherein the dominant negative variant (a)
comprises a E34A amino
acid substitution; (b) a deletion of amino acid 756; (c) a deletion of amino
acids 754-756; (d)
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a E34A arnino acid substitution and a deletion of amino acids 754-756; (e) a
deletion of
amino acids 336-756; (f) a E34A amino acid substitution and a deletion of
amino acids 336-
756; (g) a deletion of amino acids 1-500; (h) a deletion of amino acids 1-500
and a deletion
of amino acids 754-756; or (i) a deletion of amino acids =I-460 and a deletion
of amino acids
754-756, optionally wherein the dominant negative variant further comprises an
NLS
comprising the sequence KRTADGSEFESPKKKIKKV at the C terminus and/or at the N
terminus.
24. The method of claim 8, wherein the inhibitor is a dominant negative
variant of PMS2 that
inhibits the activity of PMS2, optionally wherein the dominant negative
variant comprises
one or more amino acid substitutions, insertions, and/or deletions compared to
a wild type
PMS2 protein, optionally wherein the dominant negative variant comprises (a) a
E705K
substitution, (b) a deletion of amino acids 2-607, (c) a deletion of arnino
acids 2-635, (d) a
deletion of amino acids 1-635, (e) a E41A substitution, and/or (f) a deletion
after amino acid
134 compared to a wild type PMS2 protein.
25. The method of claim 8, wherein the inhibitor is a dominant negative
variant of MSH6 that
inhibits the activity of MSH6, optionally wherein the dominant negative
variant comprises
one or more amino acid substitutions, insertions, and/or deletions compared to
a wild type
MSH6 protein, optionally wherein the dominant negative variant comprises (a) a
K1140R
substitution, and/or (b) a deletion of amino acids 2-361 compared to a wild
type MSH6
protein.
26. The method of claim 8, wherein the inhibitor comprises CDKN IA.
27. The method of claim I, wherein the prime editor comprises a napDNAbp and a
polymerase
28. The method of claim 27, wherein the napDNAbp is a nuclease active Cas9
domain, a
nuclease inactive Cas9 domain, or a Cas9 nickase domain or variant thereof.
29. The method of claim 27, wherein the napDNAbp is a Cas9 nickase comprising
one or more
amino acid substitutions in the HNH domain, optionally wherein the one or more
a.mino acid
substitutions comprises H840X, N854X, and/or N863X, wherein X is any amino
acid except
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for the original amino acid, optionally wherein the one or more amino acid
substitutions
comprises H840A, N854A, and/or N863A.
30. The method of claim 27, wherein the napDNAbp is selected from the group
consisting of:
Cas9, Cas12e, Casi2d, Cas12a, Cas12b1, Cas13a, Cas I2c, Cas12b2, Cas13a,
Casi2c,
Cas12d, Cas12e, Casi2h, Casi2i, Cas.12g, Casl2f (Cas14), Cas12f1, Cas.12j
(Cas(1:0), and
Argonaute and optionally has a nickase activity.
31. The method of claim 27, wherein the napDNAbp comprises an amino acid
sequence of any
one of SEQ ID NOs: 2, 4-67, or 104, or an amino acid sequence having at least
an 80%, 85%,
90%, 95%, or 99% sequence identity with any one of SEQ I NOs: 2,4-67, or 104.
32. The method of claim 27, wherein the napDNAbp comprises an amino acid
sequence of SEQ
ID NO: 2 or SEQ ID NO: 37 (i.e., the napDNAbp of PE1 and PE2) or an amino acid

sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with
S:EQ ID
NO: 2 or SEQ ID NO: 37.
33. The rnethod of claim 27, wherein the polyrnerase is a DNA-dependent DNA
polymerase or
an RNA-dependent DNA polymerase.
34. The method of claim 27, wherein the polymerase is a reverse transcriptase.
35. The method of claim 34, wherein the reverse transcriptase is a retroviral
reverse
transcriptase, optionally wherein the reverse transcriptase is a Moloney
Murine Leukemia
virus reverse transcriptase (MMLV-RT), optionally wherein the MMLV-RT
comprises one
or more amino acid substitutions selected from D200N, T306K, W313F, T330P, and
L603W
compared to a wild type MMLV-RT.
36. The method of claim 34, wherein the reverse transcriptase comprises an
amino acid sequence
of any one of SEQ ID NOs: 69-98 or an amino acid sequence having at least an
80%, 85%,
90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 69-98,
optionally
wherein the reverse transcriptase cotnprises an arnino acid sequence of SEQ ID
NO: 105.
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37. The rnethod of claim 27, wherein the napDNAbp and the polymerase of the
prime editor are
joined to form a fusion protein, optionally wherein the napDNAbp and the
polymerase are
joined by a linker.
38. The method of claim 37, wherein the linker comprises an amino acid
sequence of any one of
SEQ ID NOs: '102 or .118-131, or an amino acid sequence having at least an
80%, 85%, 90%,
95%, or 99% sequence identity with any one of SEQ TT) NOs: 102 or 118-131.
39. The method of claim 37, wherein the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 38, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
40. The method of claim 37, wherein the fusion protein comprises the amino
acid sequence of
SEQ ID NO: 99 or SEQ ID NO: 107, or an amino acid sequence having at least an
80%,
85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO: 99 or SEQ
ID NO:
107.
41. The method of claim 1, wherein the priine editor, the pegRNA, and the
inhibitor of the DNA
mismatch repair pathway are encoded on one or more DNA vectors.
42. The method of clai rn 41, wherein the one or more DNA. vectors comprise
AAV or lentivirus
DNA vectors.
43. The method of claim 42, wherein the AAV vector is serotype 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10.
44. The method of any one of claims 27-36, wherein the prime editor and the
inhibitor of the
DNA mism.atch repair pathway are not covalently linked.
45. The method of any one of claims 27-36, wherein the napDNAbp, the
polymerase, or the
prime editor as a fusion protein is further joined by a second linker to the
inhibitor of the
DNA mismatch repair pathway.
46. The method of claim 45, wherein the second linker comprises a self-
hydrolyzing linker,
optionally wherein the second linker i.s a T2A linker or a P2A. linker.
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47. The rnethod of claim 45, wherein the second linker comprises an arnino
acid sequence of any
one of SEQ ID NOs: 102, 118-131, or 233-236, or an amino acid sequence having
at least an
80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 102,
118-
131, or 233-236.
48. The method of claim 45, wherein the second linker is 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13,
14, 15, =16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
49. The rnethod of claim 1, wherein the one or more modifications to the
nucleic acid molecule
installed at the target site comprises one or more transitions, one or more
transversions, one
or more insertions, one or more deletions, one more inversions, or any
combination thereof,
and optionally are less than 15 bp.
50. The method of clairn 49, wherein the one or inore transitions are selected
from the group
consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
51. The method of claim 49, wherein the one or more transversions are selected
from the group
consisting of: (a) T to A; (b) 17 to G; (c) C to G; (d) C to A; (e) A. to T;
(t) A to C; (g) G to C;
and (h) G to T.
52. The method of claim 1, wherein the one or more modifications comprises
changing (1) a G:C
basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a G:C
basepair to a
C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an
A:T basepair, (6)
a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a
C:G basepair to
a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to
a T:A
basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a
C:G basepair.
53. The method of claim 1, wherein the one or more modifications comprises an
insertion or
deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or
25 nucleotides, optionally wherein the one or more edits comprises an
insertion or deletion of
1-15 nucleotides.
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54. The rnethod claim 1, wherein the one or more modifications comprises a
correction to a
mutation associated with a disease in a disease-associated gene.
55. The method of claim 54, wherein the disease-associated gene is associated
with a polygenic
disorder selected from the group consisting of: heart disease; high blood
pressure;
Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
56. The method of claim 54, wherein the disease-associated gene is associated
with a rnonogenic
disorder selected from the group consisting of: Adenosine Deaminase (ADA)
Deficiency;
Alpha-1 Anti trypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy;

Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine
Disease; Marfan
Syndrome; Neurotibromatosis Type 1; Pachyonychia Congenita; Phenylketonuria;
Severe
Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a
trinucleotide repeat disorder; a prion disease; and Tay-Sachs Disease.
57. The method of any one of claims 1-56, wherein the gRNA core comprises
minirnal sequence
homology to the sequence of the target site, optionally wherein the gRNA core
comprises no
more than 1%, 5%, 10%, 15%, 20%, 25%, or 30% sequence homology to the sequence
of the
double stranded target DNA that flanks 5, 10, 15, 20, 25, 30, 35, 40, 45, or
50 nucleotides
upstream or downstream of the position of the one or more nucleotide edits.
58. A composition for editing a nucleic acid molecule by prime editing
comprising a prime
editor, a pegRNA, and an inhibitor of the DNA mismatch repair pathway, wherein
the
composition is capable of installing one or more modifications to the nucleic
acid molecule at
a target site.
59. The composition of claim 58, wherein the composition further comprises a
second strand
nicking gRNA.
60. The composition of claim 58, wherein the prime editing efficiency is
increased by at least
1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least
3.5-fold, at least 4.0-fold,
at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at
least 6.5-fold, at least
7.0-fo1d, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least
9.0-fold, at least 9.5-fold,
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at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-fold, at
least 14-fold, at least
15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-
fold, at least 20-fold,
at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at
least 25-fold, at least
26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-
fold, at least 3 I-fold,
at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at
least 36-fold, at least
37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-
fold, at least 42-fold,
at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at
least 47-fold, at least
48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-
fold, at least 53-fold,
at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at
least 58-fold, at least
59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-
fold, at least 64-fold,
at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at
least 69-fold, at least 70-
fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold,
or at least 75-fold in
the presence of the inhibitor of the DNA mismatch repair pathway compared to
prime editing
efficiency with the prime editor and the pegRNA in the absence of the
inhibitor of the DNA
mismatch repair pathway.
61. The composition of claim 58, wherein the frequency of indel formation is
decreased by at
least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at
least 3.5-fold, at least 4.0-
fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-
fold, at least 6.5-fold, at
least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at
least 9.0-fold, at least 9.5-
fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-
fold, at least 14-fold, at
least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least
19-fold, at least 20-
fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold,
at least 25-fold, at
least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least
30-fo1d, at least 31-
fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fo1d,
at least 36-fold, at
least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least
41-fold, at least 42-
fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold,
at least 47-fold, at
least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least
52-fold, at least 53-
fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold,
at least 58-fold, at
least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least
63-fold, at least 64-
fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold,
at least 69-fold, at
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least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least
74-fold, or at least 75-
fold in the presence of the inhibitor of the DNA mismatch repair pathway
compared to the
frequency of indel formation with the prime editor and the pegRNA in the
absence of the
inhibitor of the DNA mismatch repair pathway.
62. The composition of claim 58, wherein the purity of editing outcome is
increased by at least
1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least
3.5-fold, at least 4.0-fold,
at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at
least 6.5-fold, at least
7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least
9.0-fold, at least 9.5-fold,
at least 10.0-fold, at least 11-fold, at least 12-fo1d, at least 13-fold, at
least 14-fold, at least
15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-
fold, at least 20-fold,
at least 21-fold, at least 22-fold, at least =23-fo1d, at least 24-fold, at
least 25-fold, at least
26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-
fold, at least 31-fold,
at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at
least 36-fold, at least
37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-
fold, at least 42-fold,
at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at
least 47-fold, at least
48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-
fold, at least 53-fold,
at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at
least 58-fold, at least
59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-
fold, at least 64-fold,
at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at
least 69-fold, at least 70-
fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold,
or at least 75-fold, in
the presence of the inhibitor of the DNA mismatch repair pathway compared to
the purity of
editing outcome with the prime editor and the pegRN A in the absence of the
inhibitor of the
DNA mismatch repair pathway, wherein the purity of editing outcome is measured
by the
ratio of intended edit/unintended indels.
63. The composition of claim 58, wherein the inhibitor of the DNA mismatch
repair pathway
inhibits the expression of one or more proteins of the DNA mismatch repair
pathway (MMR
proteins).
64. The composition of claim 63, wherein the one or more MMR proteins is
selected from the
group consisting of MLH1, PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3 (or
MutL
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gamma), MutS alpha (MSH2-MSI-16), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA,
RFC, EX01, POLS, and PCNA.
65. The composition of claim 63, wherein the one or more MMR proteins is MLH1.
66. The composition of claim 65, wherein MLH I comprises an amino acid
sequence of SEQ ID
NO: 204, or an amino acid sequence having at least 70%, at least 75%, at least
80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or up
to and including =100% sequence identity with SEQ ID NO: 204.
67. The composition of claim 63, wherein the inhibitor is an antibody that
inhibits the activity of
one or more proteins of the DNA mismatch repair pathway.
68. The composition of claim 63, wherein the inhibitor is a small molecule
that inhibits the
activity of the one or more proteins of the DNA mismatch repair pathway.
69. The composition of claim 63, wherein the inhibitor is a small interfering
RNA (siRNA) or a
small non-coding microRNA that inhibits the activity of the one or more
proteins of the DNA
mismatch repair pathway.
70. The composition of claim 63, wherein the inhibitor i s a dominant negative
variant of an
MMR protein that inhibits the activity of a wild type MMR protein.
71. The composition of claim 63, wherein the inhibitor is a dominant negative
valiant of MLH1
that inhibits the activity of MLHI.
72. The composition of claim 71, wherein the dominant negative valiant of
ML111 comprises one
or more amino acid substitutions, insertions, and/or deletions in an ATPase
domain compared
to a wild type MLH1 protein as set forth in SEQ JD NO: 204, wherein the one or
more amino
acid alterations impairs or abolishes ATPase activity of the dominant negative
variant of
MLH1.
73. The composition of claim 71, wherein the dominant negative valiant of MLH1
comprises one
or more amino acid substitutions, insertions, and/or deletions in an
endonuclease domain
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compared to a wild type MLH1 protein as set forth in SEQ ID NO: 204, wherein
the one or
rnore amino acid alterations impairs or abolishes endonuclease activity of the
dominant
negative variant of MLI11.
74. The composition of claim 71, wherein the dominant negative variant of MLH1
is truncated at
the C terminus compared to a wild type MLH=l protein as set forth in SEQ ID
NO: 204.
75. The composition of claim 71, wherein the dominant negative variant of
:MLH:1 is truncated at
the N terminus compared to a wild type MLHI protein as set forth in SEQ ID NO:
204.
76. The composition of any one of claims 71-75, wherein the dominant negative
variant of
MLH1 further comprises a nuclear localization signal (NLS) at the N terminus
and/or a NLS
at the C terminus.
77. The composition of claim 71, wherein the dominant negative variant is (a)
MLH1 E34A
(SEQ ID NO: 222), (b) IvILH1 A756 (SEQ ID NO: 208), (c) IVILH1 A754-756 (SEQ
ID NO:
209), (d) MLH1 E34A A754-756 (SEQ ID NO: 210), (e) MLH1 1-335 (S:EQ ID NO:
211),
(I) MLH1 1-335 E34A (SEQ ID NO: 212), (g) MLH1 1-335 NLSw4 (SEQ ID NO: 213),
(h)
ML:H1 501-756 (SEQ ID NO: 215), (i) ML:H1 501-753 (SEQ ID=NO: 216), (j) MLH1
461-
753 (SEQ ID NO: 218), or (k) NLS8v4 MLH1 501-753 (SEQ ID NO: 223), or a
polypeptide
comprising an amino acid sequence having at least 70%, at least 75%, at least
80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or up
to and including 100% sequence identity with any of SEQ ID NOs: 208-213, 215,
216, 218,
222, or 223.
78. The composition of claim 71, wherein the dominant negative variant (a)
comprises a E34A
amino acid substitution; (b) a deletion of amino acid 756; (c) a deletion of
amino acids 754-
756; (d) a E34A amino acid substitution and a deletion of amino acids 754-756;
(e) a deletion
of amino acids 336-756; (I) a E34A amino acid substitution and a deletion of
amino acids
336-756; (g) a deletion of amino acids 1-500; (h) a deletion of amino acids 1-
500 and a
deletion of amino acids 754-756; or (i) a deletion of amino acids 1-460 and a
deletion of
arnino acids 754-756, optionally wherein the dominant negative variant further
comprises an
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NES cornprising the sequence KRTADGSEFESPKKKRKV at the C terminus and/or at
the N
terminus.
79. The composition of claim 73, wherein the inhibitor is a dominant negative
variant of PMS2
that inhibits the activity of PMS2, optionally wherein the dominant negative
variant
comprises one or more amino acid substitutions, insertions, and/or deletions
compared to a
wild type PMS2 protein, optionally wherein the dominant negative variant
comprises (a) a
E705K substitution, (b) a deletion of amino acids 2-607, (c) a deletion of
amino acids 2-635,
(d) a deletion of amino acids 1-635, (e) a E41A substitution, and/or (f) a
deletion after amino
acid 134 compared to a wild type PMS2 protein.
80. The composition of claim 73, wherein the inhibitor is a dominant negative
variant of MST-16
that inhibits the activity of MSH6, optionally wherein the dominant negative
variant
comprises one or more amino acid substitutions, insertions, and/or deletions
compared to a
wild type MSH6 protein, optionally wherein the dominant negative variant
comprises (a) a
K114OR substitution, and/or (b) a deletion of amino acids 2-361 compared to a
wild type
MSH6 protein.
81. The composition of claim 73, wherein the inhibitor comprises CDKN1A.
82. The composition of claim 58, wherein the prime editor comprises a napDNAbp
and a
polymerase.
83. The composition of claim 82, wherein the napDNAbp is a nuclease active
Cas9 dornain, a
nuclease inactive Cas9 domain, or a Cas9 nickase domain or variant thereof.
84. The composition of claim 82, wherein the napDNAbp is a Cas9 nickase
comprising one or
more amino acid substitutions in the HMI domain, optionally wherein the one or
more
amino acid substitutions comprises H840X, N854X, and/or N863X, wherein X is
any amino
acid except for the original amino acid, optionally wherein the one or more
amino acid
substitutions comprises H840A, N854A, and/or N83A.
85. The composition of claim 84, wherein the napDNAbp is selected from the
group consisting
of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, ArgonauteCas12b2,
Cas13a,
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Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Casl 2f1.,
Casi 2j (Cas(1)),
and Argonaute and optionally has a nickase activity.
86. The composition of claim 84, wherein the napDNAbp comprises an amino acid
sequence of
any one of SEQ ID NOs: 2, 4-67, or PEmax or an amino acid sequence having at
least an
80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 2, 4-
67, or
I 04.
87. The composition of claim 84, wherein the napDNAbp comprises an amino acid
sequence of
SEQ ID NO: 2 or SEQ ID NO: 37 (i.e., the napDNAbp of PE1 and PE2) or an amino
acid
sequence having at least an 800A, 85%, 90%, 95%, or 99% sequence identity with
SEQ ID
NO: 2 or SEQ ID NO: 37.
88. The composition of claim 84, wherein the polymerase is a DNA-dependent DNA
polymerase
or an RNA-dependent DNA polymerase.
89. The composition of claim 84, wherein the polymerase is a reverse
transcriptase.
90. The composition of claim 89, wherein the reverse transcriptase is a
retroviral reverse
transcriptase, optionally wherein the reverse transcriptase is a Moloney
Murine Leukemia
virus reverse transcriptase (MMLV-RT), optionally wherein the MMLV-RT
comprises one
or more amino acid substitutions selected from D200N, T306K, W313F, T330P, and
L603W
compared to a wild type MMLV-RT.
91. The composition of claim 89, wherein the reverse transcriptase comprises
an amino acid
sequence of any one of SEQ ID NOs: 69-98 or an amino acid sequence having at
least an
80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 69-
98,
optionally wherein the reverse transcriptase comprises an amino acid sequence
of SEQ ID
NO: 105.
92. The composition of claim 84, wherein the napDNAbp and the polymerase of
the prime editor
a.re joined to form a fusion protein, optionally wherein the napDNAbp and the
polymerase
are joined by a linker.
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93. The composition of claim 92, wherein the linker cornprises an amino acid
sequence of any
one of SEQ ID NOs: 102, 118-131, or an amino acid sequence having at least an
80%, 85%,
90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 102, 118-131.
94. The composition of claim 92, wherein the linker is 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14,
15, 16, 17, 18, =19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
95. The composition of claim 92, wherein the fusion protein comprises the
amino acid sequence
of SEQ ID NO: 99 or SEQ ID NO: 107, or an amino acid sequence having at least
an 80%,
85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO: 99 or SEQ
ID NO:
107.
96. The composition of claim 58, wherein the prime editor, the pegRNA, and the
inhibitor of the
DNA mismatch repair pathway are encoded on one or more DNA vectors.
97. The composition of claim 96, wherein the one or more DNA vectors comprise
AAV or
lentivirus DNA vectors.
98. The composition of claim 97, wherein the AAV vector is serotype 1, 2, 3,
4, 5, 6, 7, 8, 9, or
10.
99. The composition of any one of claims 84-91, wherein the prime editor and
the inhibitor of
the DNA mismatch repair pathway are not covalently linked.
100. The composition of any one of claims 84-91, wherein the prime editor as a
fusion protein
is further joined by a second linker to the inhibitor of the DNA. mismatch
repair pathway.
101. The composition of claim 100, wherein the second linker comprises a self-
hydrolyzing
linker, optionally wherein the second linker is a T2A linker or a P2A linker.
102. The composition of claim 100, wherein the second linker comprises an
amino acid
sequence of any one of SEQ ID NOs: 102, 118-131, or 233-236, or an amino acid
sequence
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having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one
of SEQ. ID
NOs: 102, 118-131, 233-236.
103. The composition of claim 100, wherein the second linker is 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 1 I,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in
length.
104. The composition of claim 58, wherein the one or more modifications to the
nucleic acid
molecule installed at the target site comprises one or more transitions, one
or more
transversions, one or more insertions, one or more deletions, one more
inversions, or any
combination thereof, and optionally are less than 15 bp.
105. The composition of claim 104, wherein the one or more transitions are
selected from the
group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
106. The composition of claim 104, wherein the one or more transversions are
selected from
the group consisting of: (a) T to A; (b) T to G; (c) C to G; (c1) C to A; (e)
A to T; (f) A to C;
(g) G to C; and (h) G to T.
107. The composition of claim 58, wherein the one or more modifications
comprises changing
(1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair,
(3) a G:C
basepair to a C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A
basepair to an A:T
basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C
basepair, (8) a
C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an
A:T basepair
to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T
basepair to a C:G
basepair.
108. The composition of claim 58, wherein the one or more modifications
comprises an
insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, =15,
16, 17, 18, 19, 20, 21,
22, 23, 24, or 25 nucleotides, optionally wherein the one or more edits
comprises an insertion
or deletion of 1-15 nucleotides.
109. The composition claim 58, wherein the one or more modifications comprises
a correction
to a mutation associated with a disease in a disease-associated gene.
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110. The composition of claim 109, wherein the disease-associated gene is
associated with a
polygenic disorder selected from the group consisting of: heart disease; high
blood pressure;
Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
111. The composition of claim 109, wherein the disease-associated gene is
associated with a
monogenic disorder selected from the group consisting of: Adenosine :Deaminase
(ADA)
Deficiency; Alpha- l Antitrypsin Deficiency; Cystic Fibrosis; Duchenne
Muscular
Dystrophy; Galactosemia; Hemochromatosis; H:untington's Disease; Maple Syrup
Urine
Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita;
Phenylketonuria; Severe Combined immunodeficiency; Sickle Cell Disease; Smith-
Lemli-
Opitz Syndrome; a trinucleotide repeat disorder; a prion disease; and Tay-
Sachs Disease.
112. The composition of any one of claims 58-111, wherein the gRNA core
comprises
minimal sequence hornology to the sequence of the target site, optionally
wherein the gRNA
core comprises no more than 1%, 5%, 10%, 15%, 20%, 25%, or 30% sequence
honiology to
the sequence of the double stranded target DNA that flanks 5, 10, 15, 20, 25,
30, 35, 40, 45,
or 50 nucleotides upstream or downstream of the position of the one or more
nucleotide edits.
113. A polynucleotide for editing a DNA target site by prime editimr
comprising a nucleic acid
sequence encoding a napDNAbp, a polymerase, and an inhibitor of the DNA
mismatch repair
pathway, wherein the napDNAbp and polymerase is capable in the presence of a
pegRNA of
installing one or more modifications in the DNA target site.
114. The polynucleotide of claim 113, wherein the polynucleotide further
comprises a nucleic
acid sequence encoding a second strand nicking gRNA.
115. The polynucleotide of claim 113, wherein the prime editing efficiency is
increased by at
least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at
least 3.5-fold, at least 4.0-
fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-
fold, at least 6.5-fold, at
least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at
least 9.0-fold, at least 9.5-
fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-
fold, at least 14-fold, at
least 15-thld, at least 16-fold, at least 17-fold, at least 18-fold, at least
19-fold, at least 20-
fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold,
at least 25-fold, at
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least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least
30-fold, at least 31-
fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold,
at least 36-fold, at
least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least
41-fold, at least 42-
fo1d, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold,
at least 47-fold, at
least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least
52-fold, at least 53-
fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold,
at least 58-fold, at
least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least
63-fold, at least 64-
fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold,
at least 69-fold, at
least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least
74-fold, or at least 75-
fold, in the presence of the inhibitor of the DNA mismatch repair pathway.
116. The polynucleotide of claim 113, wherein the frequency of indel formation
is decreased
by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold,
at least 3.5-fold, at
least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at
least 6.0-fold, at least 6.5-
fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-
fold, at least 9.0-fold, at
least 9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at
least 13-fold, at least 14-
fold, at least 15-fold, at least 16-fold, at least 17-fo1d, at least 18-fold,
at least 19-fold, at
least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least
24-fold, at least 25-
fo1d, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold,
at least 30-fold, at
least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least
35-fold, at least 36-
fold, at least 37-fold, at least 38-fold, at least 39-fo1d, at least 40-fold,
at least 41-fold, at
least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least
46-fold, at least 47-
fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold,
at least 52-fold, at
least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least
57-fold, at least 58-
fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fo1d,
at least 63-fold, at
least 64-fold, at least 65-fo1d, at least 66-fold, at least 67-fold, at least
68-fold, at least 69-
fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold,
at least 74-fold, or at
least 75-fold, in the presence of the inhibitor of the DNA mismatch repair
pathway.
117. The polynucleotide of claim 113, wherein the inhibitor of the DNA
mismatch repair
pathway inhibits one or more proteins of the DNA mismatch repair pathway.
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118. The polynucleotide of claim 117, wherein the one or rnore proteins is
selected from the
group consisting of MLH1, PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3 (or
MutL
gamrna), MutS alpha (MSH2-MS116), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA,
RFC, EX01, POL8, and PCNA.
119. The polynucleotide of claim 117, wherein the one or more proteins is
MLH.l .
120. The polynucleotide of claim 119, wherein MLH1 comprises an amino acid
sequence of
SEQ ID NO: 204, or an amino acid sequence having at least 70%, at least 75%,
at least 80%,
at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%,
or up to and including 100% sequence identity with SEQ ID NO: 204.
121. The polynucleotide of claim 113, wherein the inhibitor is an antibody
that inhibits the
activity of one or more proteins of the DNA mismatch repair pathway.
122. The polynucleotide of claim 113, wherein the inhibitor is a small
interfering RNA
(siRNA) or a small non-coding inicro:RNA that inhibits the activity of one or
more proteins
of the DNA mismatch repair pathway.
123. The polynucleotide of claim 113, wherein the inhibitor is a dominant
negative variant of
an MMR protein that inhibits the activity of a wild type :MMR protein.
124. The polynucleotide of claim 113, wherein the inhibitor is a dominant
negative variant of
1VILH1 that inhibits M11-11.
125. The polynucleotide of claim 124, wherein the dominant negative variant is
(a) MLH1
E34A (SEQ ID NO: 222), (b) MLH I 6,756 (SEQ ID NO: 208), (c) MLFII A754-756
(SEQ
ID NO: 209), (d) :MLH:1 E34A A754-756 (SEQ ID NO: 210), (e) MLH1 1-335 (SEQ ID
NO:
211), (f) MLH1 1-335 E34A (SEQ ID NO: 212), (g) MLH1 1-335 NLSsv40 (SEQ ID NO:

213), (h) :MLH:1 501-756 (SEQ ID NO: 215), (i) :MLH:1 501-753 (SEQ ID NO:
216), (j)
MLF11 461-753 (SEQ ID NO: 218), or (k) NLS5v4 MLH1 501-753 (SEQ ID NO: 223),
or a
polypeptide comprising an amino acid sequence having at least 70%, at least
75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, at least
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99%, or up to and including 100% sequence identity with any of SEQ ID NOs: 208-
213, 215,
216, 218, 222, or 223.
126. The polynucleotide of claim 113, wherein the napDNAbp is a nuclease
active Cas9
domain, a nuclease inactive Cas9 domain, or a Cas9 nickase domain or variant
thereof.
127. The polynucleotide of claim 113, wherein the napDNAbp is selected from
the group
consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c,
ArgonauteCas12b2,
Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Casl2f (Cas14),
Casl2f1, Cas12j
(Cas(1)), and Argonaute and optionally has a nickase activity.
128. The polynucleotide of claim 113, wherein the napDNAbp cornprises an amino
acid
sequence of any one of SEQ ID NOs: 2, 4-67, or 104 or an amino acid sequence
having at
least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID
NOs: 2,4-
67, or 104.
129. The polynucleotide of claim 113, wherein the napDNAbp comprises an amino
acid
sequence of SEQ ID NO: 2 (i.e., the napDNAbp of PE1 and PE2) or an amino acid
sequence
having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID
NO: 2.
130. The polynucleotide of claim 113, wherein the polymerase is a :DNA-
dependent DNA
polymerase or an RNA-dependent DNA polymerase.
131. The polynucleotide of claim 113, wherein the polymerase is a reverse
transcriptase.
132. The polynucleotide of claim 131, wherein the reverse transcriptase
comprises an amino
acid sequence of any one of SEQ ID NOs: 69-98 or an arnino acid sequence
having at least
an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 69-
98.
133. The polynucleotide of claim 113, wherein the napDNAbp and the polymerase
of the
prime editor are joined by a linker to form a fusion protein.
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134. The polynucleotide of clairn 133, wherein the linker comprises an arnino
acid sequence of
any one of SEQ ID NOs: 102 or 118-131, or an amino acid sequence having at
least an 80%,
85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 102 or 118-
131.
135. The polynucleotide of claim 133, wherein the linker is 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12,
13, l4, 15, 16, 17, =18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38,
39, 40, 4 l, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
136. The polynucleotide of clairn 113, wherein the polynucleotide is a DNA
vector.
137. The polynucleotide of clairn 136, wherein the :DNA vector is an AAV or
lentivirus DNA
vector.
138. The polynucleotide of clairn 137, wherein the AAV vector is serotype 1,
2, 3, 4, 5, 6, 7, 8,
9, or 10.
139. The polynucleotide of claim 133, wherein the prime editor as a fusion
protein is further
joined by a second linker to the inhibitor of the DNA mismatch repair pathway.
140. The polynucleotide of claim 139, wherein the second linker comprises a
self-hydrolyzing
linker.
141. The polynucleotide of claim 139, wherein the second linker comprises an
amino acid
sequence of any one of SEQ ID NOs: 102, 118-131, or 233-236, or an amino acid
sequence
having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one
of SEQ ID NOs:
102, 118-131, or 233-236.
142. The polynucleotide of claim 139, wherein the second linker is 1, 2, 3, 4,
5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in
length.
143. The polynucleotide of claim 113, wherein the one or more modifications to
the nucleic
acid molecule installed at the target site comprises one or more transitions,
one or more
transversions, one or more insertions, one or more deletions, or one more
inversions.
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144. The polynucleotide of claim 143, wherein the one or rnore transitions are
selected from
the group consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
145. The polynucleotide of claim 143, wherein the one or more transversions
are selected from
the group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A
to T; (f) A to C; (g)
G to C; and (h) G to T.
146. A polynucleotide of claim 113, wherein the one or more modifications
comprises
changing (1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T
basepair, (3) a G:C
basepair to a C:G= basepair, (4) a T:A basepair to a G:C basepair, (5) a T: A
hasepair to an A:T
basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C
basepair, (8) a C:G
basepair to a T: A basepair, (9) a C:6 basepair to an A :T basepair, (1 0) an
A:T basepair to a T:A
basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T basepair to a
C:G basepair.
147. A polynucleotide of claim 113, wherein the one or more modifications
comprises an
insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22,
23, 24, or 25 nucleotides.
148. The polynucleotide of claim 113, wherein the one or more modifications
comprises a
correction to a disease-associated gene.
149. The polynucleotide of claim 148, wherein the disease-associated gene is
associated with a
polygenic disorder selected from the group consisting of: heart disease; high
blood pressure;
Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
150. The polynucleotide of claim 148, wherein the disease-associated gene is
associated with a
monogenic disorder selected from the group consisting of: Adenosine Deaminase
(ADA)
Deficiency; Alpha-1 Antitiypsin :Deficiency; Cystic Fibrosis; Duchenne
Muscular Dystrophy;
Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine
Disease; Marfan
Syndrome; Neurofibromatosis Type 1; :Pachyonychia Congenita; Phenylketonuria;
Severe
Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a
trinucleotide repeat disorder; a pion disease; and Tay-Sachs Disease.
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151. A cell comprising a polynucleotide of any of claims 113-150, optionally
wherein the cell
is a mammalian cell, a non-human primate cell, or a human cell.
152. A pharmaceutical composition comprising the composition of any of claims
41-80 or the
polynucleotide of any of claims 113-151, or the cell of claim 151, and a
pharmaceutical
excipient.
153. A kit comprising the composition of any of claims 58-112 or the
polynucleotide of any of
claims 113-150, a pharmaceutical excipient, and instructions for editing a DNA
target site by
pri me editing.
154. A composition comprising a first nucleic acid sequence encoding a nucleic
acid
programmable DNA binding protein (napDNAbp), a second nucleic acid sequence
encoding a
pot ymerase, and a third nucleic acid sequence encoding an inhibitor of the
DNA mismatch repair
pathway.
155. The composition of claim 154, wherein the composition further comprises a
prime
editing guide RNA (pegRNA) or a nucleic acid sequence encoding the pegRNA,
wherein the
pegRNA comprises a spacer sequence, a gRNA core, and an extension arm
comprising a DNA
synthesis template and a primer binding site (PBS), wherein the spacer
sequence comprises a
region of complementarity to a target strand of a double stranded target DNA
sequence, wherein
the gRNA core associates with the napDNAbp, wherein the DNA synthesis template
comprises a
region of complementarity to the non-target strand of the double-stranded
target DNA sequence
and one or more nucleotide edits compared to the target strand double-stranded
target DNA
sequence, and wherein the primer binding site comprises a region of
complementarity to a non-
target strand of the double-stranded target :DNA sequence.
156. The composition of claim 154, wherein the first and the second nucleic
acid sequences
are on a single polynucleotide.
157. The composition of claim 154, wherein the first, the second, and the
third nucleic acid
sequences are on a single polynucleotide.
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158. The composition of claim 157, wherein the first and the second nucleic
acid sequences
are connected to encode a napDNAbp-DNA polymerase fusion protein.
159. The composition of claim 158, wherein the first and the second nucleic
acid sequences
are connected to encode a napDNAbp-DNA polymerase fusion protein, wherein the
third nucleic
acid sequence is connected to the first or the second nucleic acide sequence
via a linker nucleic
acid sequence, optionally wherein the linker nucleic acid sequence encodes a
peptide linker,
optionally wherein the peptide linker is a self-hydrolyzing linker, optionally
wherein the self-
hydrolyzing linker is a T2A linker or a P2A linker, optionally wherein the
self-hydrolyzing
linker comprises an amino acid sequence of any one of SEQ. 1T3 NOs: 102, 118-
131, or 233-236,
or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99%
sequence identity
with any one of SEQ ID NOs: 102, 118-131, or 233-236.
160. The composition of claim 156 or 157, wherein the single polynucleotide is
part of a DNA
vector.
161. The composition of claim 156 or 157, wherein the single polynucleotide is
part of an
mRNA sequence.
162. The composition of claim 160, wherein the DNA vector is an AAV or
lentivirus DNA
vector, optionally wherein the DNA vector further comprises a promoter.
163. The composition of claim 161, wherein the mRNA sequence further comprises
a
promoter.
164. A method for editing a nucleic acid molecule by prime editing comprising:
contacting a
nucleic acid molecule with a prime editor and a prime txliting guide RNA
(pegRNA), wherein
the prime editor comprises a nucleic acid programmable DNA binding protein and
a DNA
polymerase, wherein the pegRNA a spacer sequence, a gRNA core, and an
extension arm
comprising a DNA synthesis template and a primer binding site (PBS), wherein
the DNA
synthesis template comprises three or more consecutive nucleotide mismatches
relative to the
endogenous sequence of a target site on the nucleic acid molecule, wherein the
three or more
consecutive nucleotide mismatches comprise (i) an insertion, deletion, or
substitution of x
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consecutive nucleotides that corrects a mutation (e.g., a disease associated
mutation) and (ii) an
insertion, deletion, or substitution of y consecutive nucleotides directly
adjacent to the x
consecutive nucleotides, wherein the insertion, deletion, or substitution of y
consecutive
nucleotides is a silent mutation, wherein (x y) is an integer no less than 3,
wherein y is an
integer no less than 1, and wherein inclusion of the silent mutation(s)
increases the efficiency,
reduces unintended indel frequency, and/or improves editing outcome purity by
prime editing.
165. The method of claim 164, wherein at least one of the three or more
consecutive
nucleotide mismatches results in an alteration in the amino acid sequence of a
protein expressed
from the nucleic acid molecule, and wherein at least one of the remaining
three or more
consecutive nucleotide mismatches are silent mutations.
166. The method of claim 165, wherein the silent mutations are in a coding
region of the target
nucleic acid molecule.
167. The method of claim 166, wherein the silent mutations introduce into the
nucleic acid
molecule one or more alternate codons encoding the same amino acid as the
unedited nucleic
acid molecule.
168. The method of claim 165, wherein the silent mutations are in a non-coding
region of the
target nucleic acid molecule.
169. The method of claim 168, wherein the silent rnutations do not influence
splicing, gene
regulation, RNA lifetime, or other biological properties of the target site on
the nucleic acid
molecule.
170. The method of any one of claims 164-169, wherein the DNA synthesis
template of the
pegRN=A comprises four or more, five or more, six or more, seven or more,
eight or more, nine
or more, or ten or more consecutive nucleotide mismatches relative to the
endogenous sequence
of a target site on the nucleic acid molecule.
171. The method of any one of clairns 164-170, wherein the three or more
consecutive
nucleotide mismatches evade correction by the DNA mismatch repair pathway.
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172. The m.ethod of any one of claims 164-171, wherein the prime editing
efficiency is
increased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least
3.0-fold, at least 3.5-fold,
at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at
least 6.0-fold, at least 6.5-
fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-
fold, at least 9.0-fold, at least
9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-
fo1d, at least 14-fold, at
least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least
19-fold, at least 20-
fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold,
at least 25-fold, at least
26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-
fold, at least 31-fold, at
least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least
36-fold, at least 37-
fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold,
at least 42-fold, at least
43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-
fold, at least 48-fold, at
least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least
53-fold, at least 54-
fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold,
at least 59-fold, at least
60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-
fold, at least 65-fold, at
least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least
70-fold, at least 71-fold, at
least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold
relative to a method using a
pegRNA. comprising a DNA synthesis template comprising only one consecutive
nucleotide
mismatch relative to the endogenous sequence of a target site on the nucleic
acid molecule.
173. The method of any one of claims 164-171, wherein the frequency of indel
formation is
decreased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least
3.0-fold, at least 3.5-fold,
at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at
least 6.0-fold, at least 6.5-
fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-
fold, at least 9.0-fold, at least
9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-
fold, at least 14-fold, at
least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at 1 east
l 9-fo1d, at least 20-
fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold,
at least 25-fold, at least
26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-
fold, at least 31-fold, at
least 32-fb1d, at least 33-fo1d, at least 34-fold, at least 35-fold, at least
36-fold, at least 37-
fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold,
at least 42-fold, at least
43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-
fold, at least 48-fold, at
least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least
53-fold, at least 54-
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fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold,
at least 59-fold, at least
60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-
fold, at least 65-fold, at
least 66-fold, at least 67-fold, at least 68-fold, at least 69-fold, at least
70-fold, at least 71-fold, at
least 72-fold, at least 73-fold, at least 74-fold, or at least 75-fold
relative to a method using a
pcgRNA comprising a DNA synthesis template comprising only onc consecutive
nucleotide
mismatch relative to the endogenous sequence of a target site on the nucleic
acid molecule.
174. A method for editing a nucleic acid molecule by prime editing comprising:
contacting a
nucleic acid molecule with a prime editor and a pegRNA, wherein the extension
arm of the
pegRNA comprises a DNA synthesis template comprising an insertion or deletion
of 10 or more
nucleotides relative to the endogenous sequence of a target site on the
nucleic acid molecule.
175. The method of claim 174, wherein the DNA synthesis template comprises an
insertion or
deletion of 11 or more nucleotides, 12 or rnore nucleotides, 13 or more
nucleotides, 14 or more
nucleotides, 15 or more nucleotides, 16 or more nucleotides, 17 or more
nucleotides, 18 or more
nucleotides, 19 or more nucleotides, 20 or more nucleotides, 21 or more
nucleotides, 22 or more
nucleotides, 23 or more nucleotides, 24 or more nucleotides, or 25 or more
nucleotides relative to
the endogenous sequence of a target site on the nucleic acid molecule.
176. The method of claim 175, wherein the DNA synthesis template comprises an
insertion or
deletion of 15 or more nucleotides relative to the endogenous sequence of a
target site on the
nucleic acid molecule.
177. The method of any one of claims 174-176, wherein the insertion or
deletion of 10 or
rnore nucleotides relative to the endogenous sequence of the target site on
the nucleic acid
molecule evades correction by the DNA mismatch repair pathway.
178. The method of any one of claims 174-176, wherein the prime editing
efficiency is
increased by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least
3.0-fold, at least 3.5-fold,
at least 4.0-fold, at least 4.5-fo1d, at least 5.0-fold, at least 5.5-fold, at
least 6.0-fold, at least 6.5-
fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-
fold, at least 9.0-fold, at least
9.5-fold, or at least 10.0-fold relative to a method using a pegRNA comprising
a DNA synthesis
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template comprising an insertion or deletion of fewer than 10 nucleotides
relative to the
endogenous sequence of a target site on the nucleic acid molecule.
179. The method of claim 174-176, wherein the frequency of indel formation is
decreased by
at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at
least 3.5-fold, at least 4.0-
fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-
fold, at least 6.5-fold, at least
7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least
9.0-fold, at least 9.5-fold, at
least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-fold, at
least 14-fold, at least 15-fold,
at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at
least 20-fold, at least 21-
fo1d, at least 22-fo1d, at least 23-fold, at least 24-fold, at least 25-fo1d,
at least 26-fold, at least
27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-
fold, at least 32-fold, at
least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least
37-fo1d, at least 38-
fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold,
at least 43-fold, at least
44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-
fold, at least 49-fold, at
least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least
54-fold, at least 55-
fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold,
at least 60-fold, at least
61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at =least 65-
fold, at least 66-fold, at
least 67-fold, at least 68-fold, at least 69-fold, at least 70-fold, at least
71-fold, at least 72-fold, at
least 73-fold, at least 74-fold, or at least 75-fold relative to a rnethod
using a pegRNA comprising
a DNA synthesis template comprising an insertion or deletion of fewer than 10
nucleotides
relative to the endogenous sequence of a target site on the nucleic acid
molecule.
180. A prime editing guide RNA (pegRNA) for editing a nucleic acid molecule by
prime
editing, wherein the pegRNA comprises a spacer sequence, a gRNA core, and an
extension arm
comprising a DNA synthesis template and a primer binding site (PBS), wherein
the DNA
synthesis template comprising three or more consecutive nucleotide mismatches
relative to the
endogenous sequence of a target site on the nucleic acid molecule, wherein the
three or more
consecutive nucleotide mismatches cornprise (i) an insertion, deletion, or
substitution of x
nucleotides that corrects a mutation (e.g. a disease associated mutation) and
(ii) an insertion,
deletion, or substitution of y nucleotides directly adjacent to the x
nucleotides, wherein the
insertion, deletion, or substitution of y nucleotides is a silent mutation,
wherein (x-l-y) is an
integer no less than 3, wherein y is an integer no less than 1, and wherein
inclusion of the silent
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mutation(s) increases the efficiency, reduces unintended indel frequency,
and/or improves
editing outcome purity by prime editing.
181. The pegRNA of claim 180, wherein at least one of the three or more
consecutive
nucleotide mismatches results in an alteration in the amino acid sequence of a
protein expressed
from the nucleic acid molecule, and wherein at least one of the remaining
three or more
consecutive nucleotide mismatches are silent mutations.
182. The pegRNA of claim 181, wherein the silent mutations are in a coding
region of the
target nucleic acid molecule.
183. The pegRNA of claim 182, wherein the silent mutations introduce into the
nucleic acid
molecule one or more alternate codons encoding the same amino acid as the
unedited nucleic
acid molecule.
184. The pegRNA of claim 181, wherein the silent mutations are in a non-coding
region of the
target nucleic acid molecule.
185. The pegRNA of claim 183, wherein the silent mutations are in a region of
the nucleic
acid molecule that does not influence splicing, gene regulation, RNA lifetime,
or other biological
properties of the target site on the nucleic acid molecule.
186. The pegRNA of any one of claims 180-185, wherein the extension arm of the
pegRNA
comprises four or more, five or more, six or more, seven or more, eight or
more, nine or more, or
ten or more consecutive nucleotide mismatches relative to the endogenous
sequence of a target
site on the nucleic acid molecule.
187. The pegRNA of any one of claims 180-186, wherein the three or more
consecutive
nucleotide mismatches evade the DNA mismatch repair pathway.
188. The pegRNA of any one of claims 180-186, wherein use of the pegRNA in
prime editing
results in the prime editing efficiency being increased by at least 1.5-fold,
at least 2.0 fold, at
least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at
least 4.5-fold, at least 5.0-
fold, at least 5.5-fold, at least 6.0-fo1d, at least 6.5-fold, at least 7.0-
fold, at least 7.5-fold, at least
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8.0-fo1d, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least
10.0 fold, at least 11-fold, at
least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least
16-fold, at least 17-fold,
at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at
least 22-fold, at least 23-
fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold,
at least 28-fold, at least
29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-
fold, at least 34-fold, at
least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least
39-fold, at least 40-
fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold,
at least 45-fold, at least
46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-
fold, at least 51-fold, at
least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least
56-fold, at least 57-
fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold,
at least 62-fold, at least
63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-
fold, at least 68-fold, at
least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least
73-fold, at least 74-fold, (m-
at least 75-fold relative to a pegRNA comprising a DNA synthesis template
comprising only one
consecutive nucleotide mismatch relative to the endogenous sequence of a
target site on the
nucleic acid molecule.
189. The pegRNA of any one of claims 180-186, wherein use of the pegRNA in
prime editing
results in the frequency of indel formation being decreased by at least 1.5-
fold, at least 2.0 fold,
at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at
least 4.5-fold, at least 5.0-
fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-
fold, at least 7.5-fold, at least
8.0-fo1d, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least
10.0-fold relative to a
pegRNA comprising a :DNA synthesis template comprising only one consecutive
nucleotide
mismatch relative to the endogenous sequence of a target site On the nucleic
acid molecule.
190. A prime editor system comprising the pegRNA of any one of claims 180-189
and a prime
editor, wherein the prime editor comprises a napDNAbp and a polymerase.
191. The prime editor system of claim 190, further comprising an inhibitor of
DNA mismatch
repair pathway.
192. A prime editor comprising a fusion protein comprising (i) a nucleic acid
programmable
DNA binding protein (napDNAbp) and (ii) a .DNA polymerase, wherein the
napDNAbp is a
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Cas9 nickase (nCas9) cornprising a R221K amino acid substitution, a N39K amino
acid
substitution, and an amino acid substitution that inactivates HNH domain
nuclease activity, or
corresponding amino acid substitutions thereof, relative to a wild type Cas9
as set forth in SEQ
ID NO: 2.
193. The prime editor of claim 192, wherein the nCas9 comprises a R221K, a
N39K, and a
H840A amino acid substitution compared to a wild type Cas9 as set forth in SEQ
ID NO: 2.
194. The prime editor of claim 193, wherein the nCas9 and the DNA polymerase
are
connected by a linker, optionally wherein the linker comprises the sequence of
SEQ ID NO: X5,
optionally wherein the prime editor thrther comprises a SV40 NLS at the N
terminus, optionally
wherein the prime editor further comprises a SV40 NTS and/or a c-Myc NIS at
the C terminus.
195. The prime editor of claim 192 comprising the amino acid sequence of SEQ
IT) NO: 99 or
an amino acid sequence have at least 80%, at least 85%, at least 90%, at least
95%, at least 96%,
at least 97%, at least 98%, at least 99%, or at least 100% sequence identity
with SEQ ID NO: 99.
196. A prime editor systern comprising the prime editor of any one of claims
192-195 and an
inhibitor of DNA mismatch repair pathway.
197. A prime editor system comprising the prime editor of any one of claims
192-195 and a
prime editing guide RNA (pegRNA).
198. A polynucleotide encoding the prime editor of any of claims 192-195.
199. The polynucleotide of claim 198, wherein the polynucleotide is DNA.
200. The polynucleotide of claim 198, wherein the polynucleotide is mRNA.
201. A vector comprising the polynucleotide of claim 198, optionally wherein
expression of
the fusion protein is under the control of a promoter, optionally wherein the
promoter is a U6
promoter.
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202. A prime editor systern for site specific genome modification, comprising
a (a) prime
editor comprising (i) a nucleic acid programmable DNA binding protein
(napDNAbp) and
(ii) a DNA polyrnerase, and (b) an inhibitor of the DNA mismatch repair
pathway.
203. The prime editor system of claim 202, wherein the inhibitor of the DNA
mismatch repair
pathway inhibits one or more proteins of the DNA mismatch repair pathway.
204. The prime editor system of claim 203, wherein the one or more proteins is
selected from
the group consisting of MLH1, PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3
(or MutL
gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, FCNA, RFC,
EXOI, POL8, and PCNA.
205. The prime editor system of claim 203, wherein the one or more proteins is
MiL,H1.
206. The prime editor system of claim 205, wherein the MLH1 comprises an amino
acid
sequence of SEQ ID NO: 204, or an amino acid sequence having at least 70%, at
least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at
least 99%, or up to and including 100% sequence identity with SEQ ID NO. 204.
207. The prime editor system of claim 202, wherein the inhibitor is an
antibody that inhibits
the activity of one or more proteins of the DNA mismatch repair pathway.
208. The prime editor system of claim 202, wherein the inhibitor is a small
molecule that
inhibits the activity of one or more proteins of the DNA mismatch repair
pathway.
209. The prime editor system of claim 202, wherein the inhibitor is a small
interfering RNA
(siRNA) or a small non-coding microRNA that inhibits the activity of one or
more proteins of
the :DNA mismatch repair pathway.
210. The prime editor system of claim 202, wherein the inhibitor is a dominant
negative
variant of an MIVIR protein that inhibits the activity of a wild type MMR
protein.
211. The prime editor system of claim 202, wherein the inhibitor is a dominant
negative
variant of MLH1 that inhibits MLH1.
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212. The prim.e editor system of claim 202, wherein the dominant negative
variant is (a)
MLH1 E34A (SEQ ID NO: 222), (b) MLH1 A756 (SEQ ID NO: 208), (c) MLH1 A754-756
(SEQ ID NO: 209), (d) MUM E34A A754-756 (SEQ ID NO: 210), (e) MLH1 1-335 (SEQ
ID
NO: 211), (f) MLH1 1-335 E34A (S:EQ ID NO: 212), (g) MLH1 1-335 NLS8v4O (SEQ
ID NO:
213), (h) MLH1 501-756 (SEQ ED NO: 215), (i) MLH1 501-753 (SEQ II) NO: 216),
(j) MLH1
461-753 (SEQ ID NO: 218), or (k) NL.Ssy4O MLH1 501-753 (SEQ ID NO: 223), or a
polypeptide
comprising an amino acid sequence having at least 70%, at least 75%, at least
80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or up to and
including 100% sequence identity with any of SEQ ID NOs: 208-213, 215, 216,
218, 222, or
223.
213. The prime editor system of claim 202, wherein the nap:DNAbp is a nuclease
active Cas9
domain, a nuclease inactive Cas9 domain, or a Cas9 nickase domain or variant
thereof.
214. The prime editor systeni of claiin 202, wherein the napDNAbp is selected
from the group
consisting of: Cas9, Cas12e, Casi2d, Cas12a, Cas12b1, Cas13a, Cas12c,
ArgonauteCas12b2,
Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14),
Casl2f1, Cas12j
(Cast,), and Argonaute and optionally has a nickase activity.
215. The prime editor system of claim 202, wherein the napDNAbp comprises an
amino acid
sequence of any one of SEQ ID NOs: 2, 4-67, 104 or an amino acid sequence
having at least an
80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 2, 4-
67, 104.
216. The prime editor system of claim 202, wherein the nap:DNAbp comprises an
amino acid
sequence of SEQ ID NO: 2 (i.e., the napDNAbp of PE1 and PE2) or an amino acid
sequence
having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID
NO: 2.
217. The prime editor system of claim 202, wherein the polymerase is a :DNA-
dependent DNA
polymerase or an RNA-dependent DNA polymerase.
218. The prime editor system of claim 202, wherein the polymerase is a reverse
transcriptase.
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219. The prime editor system of claim 218, wherein the reverse transcriptase
comprises an
amino acid sequence of any one of SEQ ID NOs: 69-98 or an amino acid sequence
having at
least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID
NOs: 69-98.
220. The prime editor system of claim 202, wherein the napDNAbp and the
polymerase of the
prime editor are joined by a linker to form a fusion protein, and optionally
wherein the inhibitor
is joined by a second linker to either the napDNAbp or the polymerase.
221. The prime editor system of claim 220, wherein the linker and/or second
linker comprises
an amino acid sequence of any one of SEQ ID NOs: 102 or 118-131, or an amino
acid sequence
having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one
of SEQ ID NOs:
102 or 118-131.
222. The prime editor system of claim 220, wherein the linker and/or second
linker is 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 38, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50
amino acids in length.
223. The prime editor system of claim 202, wherein the prime editor is PE1 of
SEQ ID NO:
100 or an amino acid sequence having at least 80%, at least 85%, at least 90%,
at least 95%, at
least 99%, or up to 100% sequence identity with SEQ ID NO: 100.
224. The prime editor system of claim 202, wherein the prime editor is PE2 of
SEQ ID NO:
107 or an amino acid sequence having at least 80%, at least 85%, at least 90%,
at least 95%, at
least 99%, or up to 100% sequence identity with SEQ ID NO: 107.
225. The prime editor system of claim 202, wherein the prime editor is PE1 of
SEQ ID NO:
100 or an amino acid sequence having at least 80%, at least 85%, at least 90%,
at least 95%, at
least 99%, or up to 100% sequence identity with SEQ ID NO: 100, and the
inhibitor is a
dominant negative variant of MLH1 that inhibits MLH I .
226. The prime editor system of claim 225, wherein the dominant negative
variant of MLH1 is
(a) MLII1 E34A (SEQ ID NO: 222), (b) ML/I1 A756 (SEQ ID NO: 208), (c) MLII1
A754-756
(SEQ ID NO: 209), (d) MLH1 E34A A754-756 (SEQ ID NO: 210), (e) MLH1 1-335 (SEQ
ID
=NO: 211), (1) MLH1 1-335 E34A (SEQ ID NO: 212), (g) MLIII 1-335 NI,Ssv4O (SEQ
ID NO:
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213), (h) MLHI 501-756 (SEQ ID NO: 215), (i) MLH1 501-753 (SEQ ID NO: 216),
(j) MLH1
461-753 (SEQ ID NO: 218), or (k) N'LSsv4O MLH1 501-753 (SEQ ID NO: 223), or a
polypeptide
comprising an amino acid sequence having at least 70%, at least 75%, at least
80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or up to and
including 100% sequence identity with any of SEQ ID NOs: 208-213, 215, 216,
218, 222, or
223.The prime editor system of claim 145, wherein the prime editor is PE2 of
SEQ ID NO: 107
or an amino acid sequence having at least 80%, at least 85%, at least 90%, at
least 95%õ at least
99%, or up to 100% sequence identity with SEQ ID NO: 107 and the inhibitor is
a dominant
negative variant of MLH1 that inhibits MLHI .
227. The prime editor system of claim 202, wherein the prime editor is PE2 of
SEQ ID NO:
107 or an amino acid sequence having at least 80%, at least 85%, at least 90%,
at least 95%, at
least 99%, or up to 100% sequence identity with SEQ ID NO: 107, and the
inhibitor is a
dominant negative variant of MLH1 that inhibits MLHI.
228. The prime editor system of claim 227, wherein the dominant negative
variant of MLH1 is
(a) MLH1 E34A (SEQ ID NO: 222), (b) MLH1 A756 (SEQ ID NO: 208), (c) MLH1 A754-
756
(SEQ ID NO: 209), (d) MLH1 E34A A754-756 (SEQ ID NO: 210), (e) MLE11 1-335
(SEQ ID
NO: 211), (f) MLIT1 1-335 E34A (SEQ ID NO: 212), (g) MLIII 1-335 NLSSµ14 (SEQ
ID NO:
213), (h) MLH1 501-756 (SEQ ID NO: 215), (i) MLH1 501-753 (SEQ ID NO: 216),
(j)1VILH1
461-753 (SEQ I) NO: 218), or (k)NLSsv40MLHI 501-753 (SEQ ID NO: 223), or a
polypeptide
comprising an amino acid sequence having at least 70%, at least 75%, at least
80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or up to and
including 100% sequence identity with any of SEQ ID NOs: 208-213, 215, 216,
218, 222, or
223.
229. The prime editor system of claim 202, wherein the prime editor is PE2 of
SEQ ID NO:
107 or an amino acid sequence having at least 80%, at least 85%, at least 90%,
at least 95%, at
least 99%, or up to 100% sequence identity with SEQ ID NO: 107 and the
inhibitor is a
dominant negative variant of MLHI that inhibits MLH1.
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230. The prime editor system of claim 202, wherein the DNA polymerase is a
reverse
transcriptase.
231. The prime editor system of claim 230, wherein the reverse transcriptase
is a retrovirus
reverse transcriptase.
232. The prime editor system of claim 230, wherein the reverse transcriptase
lacks RNase
activity.
233. The prime editor system of claim 230, wherein the reverse transcriptase
is a Moloney-
Murine Leukemia Virus reverse transcriptase (MMLV-RT).
234. The prime editor system of claim 233, wherein the MMLV-RT comprises an
amino acid
sequence having at least 85% identity with a sequence selected from the group
consisting of:
SEQ ID NOs: 89 and 701-716.
235. The prime editor system of claim 202, wherein the napDNAbp is a CRISPR
associated
(Cas) nuclease.
236. The prime editor system of claim 235, wherein the napDNAbp comprises a
Cas9
nuclease domain.
237. The prime editor system of claim 236, wherein the Cas9 nuclease domain is
a nickase
comprising a H840X substitution or a corresponding substitution as compared to
a wild type
Slreplococcus pyogenes Cas9 as set forth in SEQ II) NO: 18, wherein X is any
amino acid other
than histidine.
238. The prime editor system of any one of claims 202-237, further comprising
a pegRNA that
is capable of complexing with the napDNAbp of the prime editor and programming
the
napDNAbp to bind a target DNA. sequence.
239. A nucleic acid molecule encoding the prime editor system of any one of
claims 202-238,
or a component thereof.
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240. A method for precisely installing a nucleotide edit of at least 15 bp in
length in a double
stranded target DNA sequence under conditions sufficient to evade the DNA
mismatch repair
pathway, the method comprising: contacting the double stranded target DNA
sequence with a
prime editor comprising a nucleic acid programmable DNA binding protein
(napDNAbp), a
DNA polymcrasc, and a prime editing guide RNA (PEgRNA), wherein the PEgRNA
comprises
a spacer that hybridizes to a first strand of the double stranded target DNA
sequence, an
extension arm that hybridizes to a second strand of the double stranded target
DNA sequence, a
DNA synthesis template comprising the nucleotide edit, and a gRNA core that
interacts with the
napDNAbp, and wherein the PEgRNA directs the prime editor to install the
nucleotide edit in the
double stranded target DNA sequence.
241. The method of claim 240, wherein the nucleotide edit is a deletion of at
least 15 bp in
length.
242. The inethod of claim 240, wherein the nucleotide edit is an insertion of
at least 15 bp in
length.
243. The method of claim 240, wherein the nucleotide edit is a least 16 bp, 17
bp, 18 bp, 19
bp, 20 bp, 2=l bp, 22 bp, 23 bp, 24 bp, or 25 bp in length.
244. A. method for editing a nucleic acid molecule by prime editing
cornprising: contacting a
nucleic acid molecule with a prime editor and a pegRNA, thereby installing one
or more
modifications to the nucleic acid molecule at a target site, wherein the
nucleic acid molecule
is in a cell comprising a knockout of one or more genes involved in the DNA
mismatch
repair (MIVIR) pathway.
245. The method of claim 244, wherein the method further comprises contacting
the nucleic
acid molecule with a second strand nicking gRNA.
246. The method of claim 244, wherein the prirne editing efficiency is
increased by at least
1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least
3.5-fold, at least 4.0-fold,
at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at
least 6.5-fold, at least
7.0-fo1d, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least
9.0-fold, at least 9.5-fold,
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at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-fold, at
least 14-fold, at least
15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-
fold, at least 20-fold,
at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at
least 25-fold, at least
26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-
fold, at least 3 I-fold,
at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at
least 36-fold, at least
37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-
fold, at least 42-fold,
at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at
least 47-fold, at least
48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-
fold, at least 53-fold,
at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at
least 58-fold, at least
59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-
fold, at least 64-fold,
at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at
least 69-fold, at least 70-
fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold,
or at least 75-fold
relative to a method performed in a cell that does not comprise a knockout of
one or more
genes involved in NEAR.
247. The method of claim 244, wherein the frequency of indel formation is
decreased by at
least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at
least 3.5-fold, at least 4.0-
fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-
fold, at least 6.5-fold, at
least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at
least 9.0-fold, at least 9.5-
fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at least 13-
fold, at least 14-fold, at
least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least
19-fold, at least 20-
fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold,
at least 25-fold, at
least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least
30-fold, at least 31-
fo1d, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold,
at least 36-fold, at
least 37-fo1d, at least 38-fold, at least 39-fo1d, at least 40-fold, at least
41-fold, at least 42-
fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold,
at least 47-fold, at
least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least
52-fold, at least 53-
fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold,
at least 58-fold, at
least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least
63-fold, at least 64-
fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold,
at least 69-fold, at
least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at least
74-fold, or at least 75-
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fold relative to a method perforrned in a cell that does not comprise a
knockout of one or
more genes involved in MMR.
248. The method of claim 244, wherein the one or more genes involved in MMR is
selected
from the group consisting of genes encoding the proteins MLHI, PMS2 (or MutL
alpha),
PMS1 (or MutL beta), MLF1.3 (or MutL gamma), MutS alpha (MSFI2-MSH6), MutS
beta
(MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLo, and PCNA.
249. The method of claim 248, wherein the one or more genes is the gene
encoding IVILH1.
250. The method of claim 249, wherein IVILH1 comprises an amino acid sequence
of SEQ ED
NO: 204, or an amino acid sequence having at least 70%, at least 75%, at least
80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or up
to and including 100% sequence identity with SEQ ID NO: 204.
251. The method of claim 244, wherein the prime editor comprises a napDNAbp
and a
poly merase.
252. The rnethod of claim 251, wherein the napDNAbp is a nuclease active Cas9
domain, a
nuclease inactive Cas9 domain, or a Cas9 nickase domain or variant thereof.
253. The method of claim 251, wherein the napDNAbp is selected from the group
consisting
of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, Cas12b2, Cas13a,
Cas12c,
Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Casl2f1, Cas12j
(Cas(1)), and
Argonaute and optionally has a nickase activity.
254. The method of claim 251, wherein the napDNAbp comprises an amino acid
sequence of
any one of SEQ ID NOs: 2, 4-67, or 99 (PEmax) or an ainino acid sequence
having at least
an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ lD NOs: 2,
4-67, or
99 (P:Emax).
255. The method of claim 251, wherein the napDNAbp comprises an amino acid
sequence of
SEQ ID NO: 2 (i.e., the napDNAbp of PE1 and PE2) or an amino acid sequence
having at
least an 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 2.
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256. The method of claim 251, wherein the polymerase is a DNA-dependent DNA
polymerase
or an RNA-dependent DNA polymerase.
257. The method of claim 251, wherein the polymerase is a reverse
transcriptase.
258. The method of claim 257, wherein the reverse transcriptase comprises an
amino acid
sequence of any one of SEQ ID NOs: 69-98 or an amino acid sequence having at
least an
80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 69-
98.
259. The method of claim 251, wherein the napDNAhp and the polymerase of the
prime editor
are joined by a linker to form a fusion protein.
260. The method of claim 259, wherein the linker comprises an amino acid
sequence of any
one of SEQ ID NOs: 102 or 118-131, or an arnino acid sequence having at least
an 80%,
85%, 900/o, 95%, or 99% sequence identity with any one of SEQ ID NOs: 102 or
118-131.
261. The method of claim 259, wherein the linker is 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 38, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
262. The method of claim 244, wherein the one or more modifications to the
nucleic acid
molecule installed at the target site comprises one or more transitions, one
or more
transversions, one or more insertions, one or more deletions, or one more
inversions, and
optionally are less than 15 bp.
263. The method of claim 262, wherein the one or more transitions are selected
from the group
consisting of: (a) T to C; (h) A to G; (c) C to T; and (d) G to A.
264. The method of claim 262, wherein the one or more transversions are
selected from the
group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to
T; (f) A. to C; (g)
G to C; and (h) G to T.
265. The method of claim 244, wherein the one or more modifications comprises
changing (1)
a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a
G:C basepair
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to a C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to
an A:T
basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C
basepair, (8) a
C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an
A:T basepair
to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T
basepair to a C:G
bascpair.
266. The method of claim 244, wherein the one or more modifications comprises
an insertion
or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24,
or 25 nucleotides.
267. The method claim 244, wherein the one or more modifications comprises a
correction to
a disease-associated gene.
268. The method of claim 267, wherein the disease-associated gene is
associated with a
polygenic disorder selected from the group consisting of: heart disease; high
blood pressure;
Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
269. A method for editing a nucleic acid molecule by prilne editing
comprising: contacting a
nucleic acid molecule with a prime editor, a pegRNA, and an inhibitor of p53,
thereby
installing one or more modifications to the nucleic acid molecule at a target
site.
270. The method of claim 269, wherein the method further comprises contacting
the nucleic
acid molecule with a second strand nicking gRNA..
271. The method of claim 269, wherein the prime editing efficiency is
increased by at least
1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least
3.5-fold, at least 4.0-fold,
at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at
least 6.5-fold, at least
7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least
9.0-fold, at least 9.5-fold,
at least 10.0-fold, at least 11-fold, at least 12-fold, at least 13-fold, at
least 14-fold, at least
15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-
fo1d, at least 20-fold,
at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at
least 25-fold, at least
26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-
fold, at least 31-fold,
at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at
least 36-fold, at least
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37-fold, at least 38-fold, at least 39-fold, at least 40-fo1d, at least 41-
fold, at least 42-fold,
at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at
least 47-fold, at least
48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-
fold, at least 53-fold,
at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at
least 58-fold, at least
59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-
fold, at least 64-fold,
at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at
least 69-fold, at least 70-
fold, at least 71-fold, at least 72-fold, at least 73-fold, at least 74-fold,
or at least 75-fold, in
the presence of the inhibitor of p53.
272. The method of claim 269, wherein the frequency of indel formation is
decreased by at
least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at
least 3.5-fold, at least 4.0-
fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-
fold, at least 6.5-fold, at
least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at
least 9.0-fold, at least 9.5-
fold, at least 10.0-fold, at least 11-fold, at least 12-fold, at least 13-
fold, at least 14-fold, at
least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least
19-fold, at =least 20-
fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold,
at least 25-fold, at
least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least
30-fold, at least 31-
fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold,
at least 36-fold, at
least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least
41-fold, at least 42-
fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold,
at least 47-fold, at
least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least
52-fold, at least 53-
fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold,
at least 58-fold, at
least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least
63-fold, at least 64-
fold, at least 65-fold, at least 66-fold, at least 67-fold, at least 68-fold,
at least 69-fold, at
least 70-fold, at least 71-fold, at least 72-fold, at least 73-fo1d, at least
74-fold, or at least 75-
fold, in the presence of the inhibitor of p53.
273. The method of claim 269, wherein the inhibitor of p53 is a protein.
274. The method of claim 273, wherein the protein is 153.
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275. The method of claim 269, wherein the inhibitor of p53 is an antibody that
inhibits the
activity of p53.
276. The method of claim 269, wherein the inhibitor of p53 is a small molecule
that inhibits
the activity of p53.
277. The method of claim 269, wherein the inhibitor of p53 is a small
interfering RNA
(siRNA) or a small non-coding micro:RNA that inhibits the activity of p53.
278. The method of claim 269, wherein the prime editor comprises a napDNAbp
and a
polymerase.
279. The method of claim 278, wherein the napDNAbp is a nuclease active Cas9
domain, a
nuclease inactive Cas9 domain, or a Cas9 nickase domain or variant thereof
280. The method of claim 278, wherein the napDNAbp is selected from the group
consisting
of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas13a, Cas12c, Cas12b2, Cas13a,
Cas12c,
Cas12d, Cas12e, Cas12h, Casi2i, Cas12g, Cas12f (Cas14), Casl2f1, Cas12j
(Cas(1)), and
Argonaute and optionally has a nickase activity.
281. The method of claim 278, wherein the napDNAbp comprises an amino acid
sequence of
any one of SEQ ID NOs: 2, 4-67, or 104, or an amino acid sequence having at
least an 80%,
85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 2, 4-67,
or 104.
282. The method of claim 278, wherein the napDNAbp comprises an amino acid
sequence of
SEQ JD NO: 2 or SEQ ID NO: 37 (i.e., the napDNAbp of PE1 and PE2) or an amino
acid
sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence identity witb
SEQ ID
=NO: 2 or SEQ ID NO: 37.
283. The method of claim 278, wherein the polymerase is a DNA-dependent DNA
polymerase
or an RNA-dependent DNA polymerase.
284. The method of claim 278, wherein the polymerase is a reverse
transcriptase.
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285. The method of claim 284, wherein the reverse transcriptase comprises an
amino acid
sequence of any one of SEQ ID NOs: 69-98 or an amino acid sequence having at
least an
80%, 85 A, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 69-
98.
286. The method of claim 278, wherein the napDNAbp and the polymerase of the
prime editor
are joined by a linker to form a fusion protein.
287. The method of claim 286, wherein the linker comprises an amino acid
sequence of any
one of SEQ ID NOs: 102 or 118-131, or an amino acid sequence having at least
an 80%,
85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NOs: 102 or 118-
131
288. The method of claim 286, wherein the linker is 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 38, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
289. The method of claim 269, wherein the prime editor, the pegRNA, and the
inhibitor of p53
are encoded on one or more DNA vectors.
290. The method of claim 289, wherein the one or more DNA vectors comprise AAV
or
lentivirus DNA vectors.
291. The method of claim 290, wherein the AAV vector is serotype 1, 2, 3, 4,
5, 6, 7, 8, 9, or
10.
292. The method of claim 286, wherein the prime editor as a fusion protein is
further joined by
a second linker to the inhibitor of p53.
293. The method of claim 292, wherein the second linker comprises a self-
hydrolyzing linker.
294. The method of claim 292, wherein the second linker comprises an amino
acid sequence
of any one of SEQ ID NOs: 102, 118-131, or 233-236, or an amino acid sequence
having at
least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one of S:EQ ID
NOs: 102,
118-131, or 233-236.
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295. The method of claim 292, wherein the second linker is 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
296. The method of claim 269, wherein the one or more modifications to the
nucleic acid
molecule installed at the target site comprises one or more transitions, one
or more
transversions, one or more insertions, one or more deletions, or one more
inversions, and
optionally wherein the one or more modifications are less than 15 bp.
297. The method of claim 296, wherein the one or more transitions are selected
from the group
consisting of: (a) T to C; (b) A to G; (c) C to T; and (d) G to A.
298. The method of claim 296, wherein the one or more transversions are
selected from the
group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to
T; (f) A to C; (g)
G to C; and (h) G to T.
299. The method of claim 269, wherein the one or more inodifications comprises
changing (1)
a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair, (3) a
G:C basepair
to a C:G basepair, (4) a '11: A basepair to a G:C basepair, (5) a 'I':A
basepair to an A:T
basepair, (6) a T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C
basepair, (8) a
C:G basepair to a T:A basepair, (9) a C:G basepair to an A:T basepair, (10) an
A:T basepair
to a T:A basepair, (11) an A:T basepair to a G:C basepair, or (12) an A:T
basepair to a C:G
basepair.
300. The method of claim 269, wherein the one or more modifications comprises
an insertion
or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24,
or 25 nucleotides.
301. The method claim 269, wherein the one or more modifications comprises a
correction to
a disease-associated gene.
302. The method of claim 301, wherein the disease-associated gene is
associated with a
polygenic disorder selected from the group consisting of: heart disease, high
blood pressure,
Alzheimer's disease, arthritis, diabetes, cancer, and obesity.
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303. The method of claim 301, wherein the disease-associated gene is
associated with a
monogenic disorder selected from the group consisting of: Adenosine Deaminase
(ADA)
Deficiency, Alpha-1 Antitrypsin Deficiency, Cystic Fibrosis, Duchenne Muscular
Dystrophy,
Galactosemia, Hemochromatosis, Huntington's Disease, :Maple Syrup Urine
Disease, Marfan
Syndrome, Neurofibromatosis Type 1, Pachyonychia Congcnita, Phenylketonuria,
Severe
Combined Immunodeficiency, Sickle Cell Disease, Smith-Lemli-Opitz Syndrome, a
trinucleotide repeat disorder, a prion disease, and Tay-Sachs Disease.
304. The method of any of the preceding claims, wherein the nucleic acid
molecule is in a cell.
305. The method of claim 304, wherein the cell is a mammalian cell, a non-
human primate
cell, or a human cell.
306. The method of claim 304, wherein the cell is ex vivo.
307. The method of claim 304, wherein the cell is in a subject, optionally
wherein the subject
is human.
308. A method for treating a disease in a subject in need thereof, the method
comprising
administering to the subject: (i) a prime editor, (ii) a pegRN=A, and (iii) an
inhibitor of a DNA
mismatch repair pathway, wherein the prime editor comprises a nucleic acid
programmable
DNA binding protein (napDNAbp) and a DNA polymerase,
wherein the pegRNA comprises a spacer sequence, a gRNA core, and an extension
arrn
comprising a DNA synthesis template and a primer binding site (PBS),
wherein the spacer sequence comprises a region of complementarity to a target
strand of a
double stranded target DNA sequence in the subject,
wherein the gRNA core associates with the napDNAbp,
wherein the DNA synthesis template comprises a region of complementarity to
the non-target
strand of the double-stranded target :DNA sequence and one or more nucleotide
edits
compared to the target strand double-stranded target DNA sequence;
wherein the primer binding site comprises a region of complementarity to a non-
target strand
of the double-stranded target DNA sequence,
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wherein the prime editor and the pegRNA install the one or more nucleotide
edits in the
double stranded target DNA, wherein installation of the one or more nucleotide
edits corrects
one or more mutations in the double stranded target DNA associated with the
disease,
thereby treating the disease in the subject.
309. A method for treat a disease in a subject in need thereof, the method
comprising
administering to the subject: (i) a prime editor and (ii) a pegRNA wherein the
prime editor
comprises a nucleic acid programmable DNA binding protein (napDNAbp) and a DNA

polymerase,
wherein the pegRNA comprises a spacer sequence, a gRNA core, and an extension
arm
comprising a DNA synthesis template and a primer binding site (PBS),
wherein the spacer sequence comprises a region of complementarity to a target
strand of a
double stranded target DNA sequence in the subject,
wherein the gRNA core associates with the napDNAbp,
wherein the DNA synthesis template comprises a region of complementarity to
the non-target
strand of the double-stranded target DNA sequence and comprises three or more
consecutive
nucleotide mismatches relative to the endogenous sequence of the double
stranded target
DNA sequence, wherein the three or more consecutive nucleotide mismatches
comprise (i)
an insertion, deletion, or substitution of x nucleotides that corrects a
mutation (e.g. a disease
associated mutation) and (ii) an insertion, deletion, or substitution of y
nucleotides directly
adjacent to the x nucleotides, wherein the insertion, deletion, or
substitution of y nucleotides
is a silent mutation, wherein (x+y) is an integer no less than 3, wherein y is
an integer no less
than 1, and wherein inclusion of the silent mutation(s) increases the
efficiency, reduces
unintended indel frequency, and/or improves editing outcome purity by prime
editing,
wherein the primer binding site comprises a region of complementarity to a non-
target strand
of the double-stranded target DNA sequence,
wherein the prime editor and the pegRNA install the insertion, deletion, or
substitution of x
nucleotides in the double stranded target DNA, wherein installation insertion,
deletion, or
substitution of x nucleotides corrects one or more mutations in the double
stranded target
DNA associated with the disease, thereby treating the disease in the subject.
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310. The method of claim 308 or 309, wherein the subject is a human.
311. A fusion protein comprising a nucleic acid programmable DNA binding
protein
(napDNAbp) domain and a domain comprising an RNA-dependent DNA polymerase
activity, wherein the fusion protein comprises an amino acid sequence of SEQ
ID NO: 99, or
an amino acid sequence have at least 80%, at least 85%, at least 90%, at least
95%, at least
96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence
identity with SEQ
ID NO: 99
312. A fusion protein comprising a nucleic acid programmable DNA binding
protein
(napDNAbp) domain and a domain comprising an RNA-dependent DNA polyrnerase
activity, wherein the napDNAbp comprises an amino acid sequence of SEQ lD NO:
104, or
an amino acid sequence have at least 80%, at least 85%, at least 90%, at least
95%, at least
96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence
identity with SEQ
ID NO: 104.
313. A fusion protein comprising a nucleic acid programmable DNA binding
protein
(napDNAbp) domain and a domain comprising an RNA-dependent DNA polymerase
activity, wherein the domain comprising an RNA-dependent DNA polymerase
activity
comprises an amino acid sequence of SEQ ID NO: 98, or an amino acid sequence
have at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at
least 99%, or at least 100% sequence identity with SEQ JD NO: 98.
314. The fusion protein of any of claims 311-313, further comprising a linker
that joins the
napDNAbp and the domain comprising the RNA-dependent :DNA polymerase activity.
315. The fusion protein of claim 314, wherein the linker comprises SEQ ID NO:
105.
316. The fusion protein of claim 311, wherein the napDNAbp is a Cas9 nickase.
317. The fusion protein of claim 316, wherein the Cas9 nickase comprises an
H840A
substitution and at least one substitution at R221 or N394 relative to SEQ ID
NO: 37.
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318. A complex comprising a fusion protein of any one of claims 311-317 and a
PEgRNA,
wherein the PEgRNA directs the fusion protein to a target DNA sequence for
prime editing.
319. The complex of claim 318, wherein the PEgRNA comprises a guide RNA and a
nucleic
acid extension arm at the 3' or 5' end of the guide RNA.
320. The complex of claim 319, wherein the PEgRNA is capable of binding to a
napDNAbp
and directing the napDNAbp to the target DNA sequence.
321. A polynucleotide encoding the fusion protein of any of claims 311-317.
322. A. vector comprising the polynucleotide of claim 321, wherein expression
of the fusion
protein is under the control of a promoter.
323. The vector of claim 322, wherein the promoter is a U6 promoter.
324. A cell comprising the fusion protein of any of claims 311-317 and a
PEgRNA bound to
the napDNAbp of the fusion protein.
325. A cell comprising a complex of any one of claims 318-320.
326. A pharmaceutical composition comprising: (i) a fusion protein of any of
claims 311-317,
the complex of claims 318-320, the polynucleotide of claim 321, or the vector
of claims 322-
323; and (ii) a pharmaceutically acceptable excipient.
327. A method for editing a nucleic acid molecule by prime editing comprising:
contacting a
nucleic acid molecule with a modified prime editor of any one of claims 311-
317 and a
pegRNA, thereby installing one or more modifications to the nucleic acid
molecule at a
target site.
328. The method of claim 327, wherein the method further comprises contacting
the nucleic
acid molecule with a second strand nicking gRNA.
329. The method of claim 327, wherein the prime editing efficiency is
increased by at least
1.5-fold, at least 2.0 fold, at least 2.5-fold, at least 3.0-fold, at least
3.5-fold, at least 4.0-fo1d,
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at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at
least 6.5-fold, at least
7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least
9.0-fold, at least 9.5-fold,
or at least 10.0 fold relative to prime editing with PE2.
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Description

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 7
CONTENANT LES PAGES 1 A 132
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 7
CONTAINING PAGES 1 TO 132
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:

WO 2022/150790 PCT/US2022/012054
PRIME EDITOR VARIANTS, CONSTRUCTS, AND METHODS FOR ENHANCING
PRIME EDITING EFFICIENCY AND PRECISION
GOVERNMENT SUPPORT
[1] This invention was made with government support under Grant Nos.
AI142756,
All 50551, HG009490, EB022376, EB031172, GM118062, CA072720, GM138167,
U01AI142756, RM1HG009490, R01EB022376, and R35GM118062 awarded by the National

Institutes of Health, and Grant No. EIR0011-17-2-0049 awarded by the
Department of
Defense. The government has certain rights in the invention.
RELATED APPLICATIONS
[2] This application claims the priority under 35 U.S.C. 119(e) to U.S.
Provisional
Application U.S.S.N. 63/255,897, filed October 14, 2021, U.S. Provisional
Application U.S.S.N.
63/231,230, filed August 9, 2021, U.S. Provisional Application U.S.S.N.
63/194,913, filed May
28, 2021, U.S. Provisional Application U.S.S.N. 63/194,865, filed May 28,
2021, U.S.
Provisional Application U.S.S.N. 63/176,202, filed April 16, 2021, and U.S.
Provisional
Application U.S.S.N. 63/136,194, filed January 11, 2021, each of which is
incorporated herein
by reference.
INCORPORATION BY REFERENCE
131 In addition, this application refers to and incorporates by reference
the entire contents of
each of the following patent applications directed to prime editing previously
filed by one or
more of the present inventors: U.S. Provisional Application U.S.S.N.
62/820,813, filed March
19, 2019; U.S. Provisional Application U.S.S.N. 62/858,958, filed June 7,
2019; U.S. Provisional
Application U.S.S.N. 62/889,996, filed August 21, 2019; U.S. Provisional
Application U.S.S.N.
62/922,654, filed August 21, 2019; U.S. Provisional Application U.S.S.N.
62/913,553, filed
October 10, 2019; U.S. Provisional Application U.S.S.N. 62/973,558, filed
October 10, 2019;
U.S. Provisional Application U.S.S.N. 62/931,195, filed November 5, 2019; U.S.
Provisional
Application U.S.S.N. 62/944,231, filed December 5, 2019; U.S. Provisional
Application
U.S.S.N. 62/974,537, filed December 5, 2019; U.S. Provisional Application
U.S.S.N.
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WO 2022/150790 PCT/US2022/012054
62/991,069, filed March 17, 2020; U.S. Provisional Application U.S.S.N.
63/100,548, filed
March 17, 2020; International PCT Application No. PCT/US2020/023721, filed
March 19, 2020;
International PCT Application No. PCT/US2020/023553, filed March 19, 2020;
International
PCT Application No. PCT/US2020/023583, filed March 19, 2020; International PCT

Application No. PCT/US2020/023730, filed March 19, 2020; International PCT
Application No.
PCT/US2020/023713, filed March 19, 2020; International PCT Application No.
PCT/US2020/023712, filed March 19, 2020; International PCT Application No.
PCT/US2020/023727, filed March 19, 2020; International PCT Application No.
PCT/US2020/023724, filed March 19, 2020; International PCT Application No.
PCT/US2020/023725, filed March 19, 2020; International PCT Application No.
PCT/US2020/023728, filed March 19, 2020; International PCT Application No.
PCT/US2020/023732, filed March 19, 2020; and International PCT Application No.

PCT/US2020/023723, filed March 19, 2020.
BACKGROUND OF THE INVENTION
[4] The recent development of prime editing enables the insertion,
deletion, and/or
replacement of genomic DNA sequences without requiring error-prone double-
strand DNA
breaks. See Anzalone et at,"Search-and-replace genome editing without double-
strand breaks or
donor DNA," Nature, 2019, Vol.576, pp. 149-157, the contents of which are
incorporated herein
by reference. Prime editing uses an engineered Cas9 nickase-reverse
transcriptase fusion protein
(e.g., PE1 or PE2) paired with an engineered prime editing guide RNA (pegRNA)
that not only
directs Cas9 to a target genomic site, but also which encodes the information
for installing the
desired edit. Without wishing to be bound by any particular theory, prime
editing proceeds
through a presumed multi-step editing process: 1) the Cas9 domain binds and
nicks the target
genomic DNA site, which is specified by the pegRNA's spacer sequence; 2) the
reverse
transcriptase domain uses the nicked genomic DNA as a primer to initiate the
synthesis of an
edited DNA strand using an engineered extension on the pegRNA as a template
for reverse
transcription-this generates a single-stranded 3' flap containing the edited
DNA sequence; 3)
cellular DNA repair resolves the 3' flap intermediate by the displacement of a
5' flap species that
occurs via invasion by the edited 3' flap, excision of the 5' flap containing
the original DNA
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sequence, and ligation of the new 3' flap to incorporate the edited DNA
strand, forming a
heteroduplex of one edited and one unedited strand; and 4) cellular DNA repair
replaces the
unedited strand within the heteroduplex using the edited strand as a template
for repair,
completing the editing process.
151 Since 2019, prime editing has been applied to introduce genetic changes
in a wide variety
of cells and/or organisms. Given its rapid adoption, prime editing represents
a powerful tool for
genomic editing. Despite its versatility and wide-scale use, prime editing
efficiency can vary
widely across different edit classes, target loci, and cell types (Anzalone
etal., 2019). Thus,
modifications to prime editing systems which result in increasing the
specificity and/or
efficiency of the prime editing process would significantly help advance the
art. In particular,
modifications that facilitate more efficient incorporation of the edited DNA
strand synthesized
by the prime editor into the target genomic site are desirable. It is also
desirable to reduce the
frequency of indel byproducts that can form as a result of prime editing. Such
further
modifications to prime editing would advance the art.
SUMMARY OF THE INVENTION
161 In one aspect, the present disclosure relates to the observation that
the efficiency and/or
specificity of prime editing is impacted by a cell's own DNA mismatch repair
(MMR) DNA
repair pathway. MMR is a multi-factor pathway that is involved in correcting
basepair
mismatches and insertion/deletion mispairs generated during DNA replication
and
recombination. As described herein, the inventors developed a novel genetic
screening
method¨referred to in one embodiment as "pooled CRISPRi screen for prime
editing
outcomes"¨which led to the identification of various genetic determinates,
including MMR, as
affecting the efficiency and/or specificity of prime editing. Accordingly, in
one aspect, the
present disclosure provides novel prime editing systems comprising a means for
inhibiting and/or
evade the effects of MMR, thereby increasing the efficiency and/or specificity
of prime editing.
In one embodiment, the disclosure provides a prime editing system that
comprises an MMR-
inhibiting protein, such as, but not limited to, a dominant negative variant
of an MMR protein,
such as a dominant negative MLI-11 protein (i.e., "MLIIIdn"). In another
embodiment, the prime
editing system comprises the installation of one or more silent mutations
nearby an intended edit,
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thereby allowing the intended edit from evading MMR recognition, even in the
absence of an
1M/1R-inhibiting protein, such as an MtHldn. In another aspect, the disclosure
provides a novel
genetic screen for identifying genetic determinants, such as MMR, that impact
the efficiency
and/or specificity of prime editing. In still further aspects, the disclosure
provides nucleic acid
constructs encoding the improved prime editing systems described herein. The
disclosure in
other aspects also provides vectors (e.g., AAV or lentivirus vectors)
comprising nucleic acids
encoding the improved prime editing system described herein. In still other
aspects, the
disclosure provides cells comprising the improved prime editing systems
described herein. The
disclosure also provides in other aspects the components of the genetic
screens, including nucleic
acid and/or vector constructs, guide .RNA, pegRNAs, cells (e.g., CRISPRi
cells), and other
reagents and/or materials for conducting the herein disclosed genetic screens.
In still other
aspects, the disclosure provides compositions and kits, e.g., pharmaceutical
compositions,
comprising the improved prime editing system described herein and which are
capable of being
administered to a cell, tissue, or organism by any suitable means, such as by
gene therapy,
mRNA delivery, virus-like particle delivery, or ribonucleoprotein (RNP)
delivery. In yet another
aspect, the present disclosure provides methods of using the improved prime
editing system to
install one or more edits in a target nucleic acid molecule, e.g., a genomic
locus. In still another
aspect, the present disclosure provides methods of treating a disease or
disorder using the
improved prime editing system to correct or otherwise repair one or more
genetic changes (e.g., a
single nucleotide polymorphism) in a target nucleic acid molecule, e.g., a
genomic locus
comprising one or more disease-causing mutations.
171 Thus, in various aspects, the present disclosure describes an improved
and modified
approach to prime editing that comprises inhibiting the DNA mismatch repair
(MMR) system
during prime editing. The inventors have surprisingly found that the editing
efficiency of prime
editing may be significantly increased (e.g., at least a 2-fold increase, at
least a 3-fold increase, at
least a 4-fold increase, at least a 5-fold increase, at least a 6-fold
increase, at least a 7-fold
increase, at least an 8-fold increase, at least a 9-fold increase, at least a
10-fold increase, or more)
when one or more functions of the DNA mismatch repair (MMR) system are
inhibited, blocked,
or otherwise inactivated during prime editing (such as using the MLHldn
inhibitor of MMR). In
addition, the inventors have surprisingly found that the frequency of indel
formation resulting
from prime editing may be significantly decreased (e.g., about a 2-fold
decrease, about a 3-fold
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decrease, about a 4-fold decrease, about a 5-fold decrease, about a 6-fold
decrease, about a 7-fold
decrease, about a 8-fold decrease, about a 9-fold decrease, or about a 10-fold
decrease or lower)
when one or more functions of the DNA mismatch repair (MMR) system are
inhibited, blocked,
or otherwise inactivated during prime editing.
E81 The present disclosure also describes in other embodiments an improved
and modified
approach to prime editing that comprises evading the DNA mismatch repair (MMR)
system
during prime editing. The inventors have surprisingly found that the editing
efficiency of prime
editing may be significantly increased (e.g., at least 1.5-fold, at least 2.0-
fold, at least 2.5-fold, at
least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at
least 5.0-fold, at least 5.5-
fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-
fold, at least 8.0-fold, at least
8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0-fold, at least
11-fold, at least 12-fold, at
least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least
17-fold, at least 18-
fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold,
at least 23-fold, at least
24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-
fold, at least 29-fold, at
least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least
34-fold, at least 35-
fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold,
at least 40-fold, at least
41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-
fold, at least 46-fold, at
least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least
51-fold, at least 52-
fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold,
at least 57-fold, at least
58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-
fold, at least 63-fold, at
least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least
68-fold, at least 69-fold,
at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at
least 74-fold, or at least 75-
fold increased) when one or more silent mutations are installed nearby a
desired site for
installing a genetic change by prime editing, in the presence or absence of an
inhibitor of IMMR.
In addition, the inventors have surprisingly found that the frequency of indel
formation resulting
from prime editing may be significantly decreased (e.g., at least 1.5-fold, at
least 2.0-fold, at least
2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least
4.5-fold, at least 5.0-fold, at
least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at
least 7.5-fold, at least 8.0-
fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0-
fold, at least 11-fold, at least
12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-
fold, at least 17-fold, at
least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least
22-fold, at least 23-
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fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold,
at least 28-fold, at least
29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-
fold, at least 34-fold, at
least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least
39-fold, at least 40-
fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold,
at least 45-fold, at least
46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-
fold, at least 51-fold, at
least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least
56-fold, at least 57-
fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold,
at least 62-fold, at least
63-fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-
fold, at least 68-fold, at
least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least
73-fold, at least 74-fold, or
at least 75-fold decreased) when one or more silent mutations are installed
nearby a desired site
for installing a genetic change by prime editing, in the presence or absence
of an inhibitor of
MMR.
[9] In some embodiments, the disclosure describes an improved prime editing
system
referred to herein as "PE4," which includes PE2 plus an MLH1 dominant negative
protein (e.g.,
wild-type MLH1 with amino acids 754-756 truncated as described further
herein). In certain
embodiments, the MLHIdn is expressed in trans in a cell comprising the PE2
fusion protein.
The MLFIldn and the PE2 may be provided together or separate, e.g., by
delivery on separate
plasmids, separate vectors (e.g., AAV or lentivirus vectors), separate vector-
like particles,
separate ribonucleoprotein complexes (RNPs), or by delivery on the same
plasmids, same
vectors (e.g., AAV or lentivirus vectors), same vector-like particles, same
ribonucleoprotein
complexes (RNPs). In other embodiments, the MLHldn may be fused to PE2 or
otherwise
associated with, coupled, or joined to PE2 such that they are co-delivered.
11.01 In other embodiments, the disclosure describes an improved prime editing
system
referred to as "PIES," which includes IPE3 (which is PE2 plus a second-strand
nicking guide
RNA) plus an MLH1 dominant negative protein (e.g., wild-type MLH1 with amino
acids 754-
756 truncated as described further herein). In certain embodiments, the MLHldn
is expressed in
trans in a cell comprising the PE3 prime editor. The MLHIdn and the PE3 may be
provide
together or separate, e.g., by delivery on separate plasmids, separate vectors
(e.g., AAV or
lentivirus vectors), separate vector-like particles, separate
ribonucleoprotein complexes (RNPs),
or by delivery on the same plasmid, same vector (e.g., AAV or lentivirus
vectors), same vector-
like particles, same ribonucleoprotein complexes (RNPs). In other embodiments,
the MLHldn
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may be fused to PE3 or otherwise associated with, coupled, or joined to PE3
such that they are
co-delivered.
[111 In other aspects, the present disclosure describes an optimized PE2 prime
editor
architecture referred to herein as "PEmax." PEmax is a modified form of PE2
which comprises
modified reverse transcriptase codon usage, SpCas9 mutations, NLS sequences,
and is described
in FIG. 54B. Specifically, PEmax refers to a PE complex comprising a fusion
protein
comprising Cas9 (R221K N394K H840A) and a variant MMLV RT pentamutant (D200N
T306K W313F T330P L603W) having the following structure: [bipartite NLS]-
[Cas9(R221K)(N394K)(H840A)MlinkerHIVIMLV_RT(D200N)(T330P)(1,603W)]-1bipartite
NLSHNLS] + a desired PEgRNA, wherein the PE fusion has the amino acid sequence
of SEQ
ID NO: 99, which is shown as follows:
MKRTADGSEFESPKKKRKVDICKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNT
DRHSIKILNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQE1FSNEMAKVD
DSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYP1TYHLRKKLVDS17DKADL
RLIYLALAHMEICFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA
KAILSARLSKSRKLENLIAQLPGEKKAGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ
LSKDTYDDDILDNILAQIGIDQYADILFLAAKNLSDAILLSDILRVNTEITKAPLSASMIK
RYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPI
LEKMDGTEELLVKLKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD
NREKIEKILTFRIPYYVGPLA.RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFI
ERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKA
IVDLLFKTNRK'VTVKQLKEDYFKKIECIFIDSVEISGVE:DRFNASLGTYH:DLLKIIKDIK
DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGW
GRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLLHDDSLTFKEDIQKAQVSGQ
GDSLHEIHANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQK
GQKNSRERM1CRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE
LDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYW
RQLLNAKLITQRKF.DN.LTKAERGGLSE.LDKAGFIKRQLVETRQITKHVAQILDSRM
NTKYDENDKLIREVKVITLKSKLVSDITIKDFQFYKVREINNYHHAHDAYLNAVVGT
ALIKKYPKLESEFVYGD'YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL
ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSNIPQVNIVICKTEVQTGGFSKE
SILPKRNSDKLIARKICDWDPKKYGGFDSPTVAYSVINVAKVEKGKSKKLKSVICEL
LGITIMERSSFEKNPMFLEAKGYKEVKXDLILKLPIKYSLFELENGRKRMLASAGEL
QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIEEQISEFS
KRVILADANLDKVLSAYNKHRDKPIREQAENHHLFTLTNLGAPAAFKNIFDTTIDRK
RYTSTKEVLDATLIHQSITGLYETRIDLSQLGG.DSGGSSGGSKRTA.DGSEFESPKKK
RKVSGGSSGGSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP
LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTND
YRPVQDLREVNKRVEDIEIPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPL
FAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQIIPDLILLQYVDDLL
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LAAISELDCQQGTRALLQTLGNLGYRA.SAKKAQICQKQVKYLGYLLKEGQRWLTEAR
KETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKA
YQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDP
VAAGWPPCLRMVAAIAVLIKDAGKLTMGQPINILAPHA'VEALVKQPPDRWLSNARMI
HYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDA
DHTWYTDGSSLLQEGQRKAGAAVETETEVIWAKALPAGISAQRAELIALTQALKMAEG
KKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSBHCP
GHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFESPKK
KRKVGSGPAAKRVKID (SEQ ID NO: 99)
KEY:
BIPARTITE SV40 NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP: (SEQ ID NO:
101), BOTTOM: (SEQ ED NO: 140)
CAS9(R221K N39K I1840A) (SEQ ID NO: 104)
SGGSX2-BIPARTITE SV4ONLS-SGGSX2 LINKER (SEQ ID NO: 105)
M-MLV reverse transcriptase (D200N T306K W313F T330P L603W) (SEQ ID NO: 98)
Other linker sequence (SEQ ID NO: 122)
Other linker sequence (SEQ ID NO: 106)
c-Myc NLS PAAKRVKLD (SEQ ID NO: 135)
[121 In some embodiments, the PE4 may be modified to substitute the PE2 fusion
protein with
PEmax. In such cases, the modified prime editing system may be referred to as
"PE4max."
[I.3] In some embodiments, the PE5 may be modified to substitute the PE3 prime
editor with
PEmax. In such cases, the modified prime editing system may be referred to as
"PE5max" and
includes a second stranding nicking guide RNA.
[141 The inventors developed prime editing which enables the insertion,
deletion, and/or
replacement of genomic DNA sequences without requiring error-prone double-
strand DNA
breaks. The present disclosure now provides an improved method of prime
editing involving the
blocking, inhibiting, evading, or inactivation of the MMR pathway (e.g., by
inhibiting, blocking,
or inactivating an MMR pathway protein, including MUM ) during prime editing,
whereby doing
so surprisingly results in increased editing efficiency and reduced indel
formation. As used
herein, "during" prime editing can embrace any suitable sequence of events,
such that the prime
editing step can be applied before, at the same time, or after the step of
blocking, inhibiting,
evading, or inactivating the MMR pathway (e.g., by targeting the inhibition of
MLH1).
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11.51 In various aspects and without wishing to be bound by any particular
theory, prime
editing uses an engineered Cas9 nickase¨reverse transcriptase fusion protein
(e.g., PE1 or PE2)
paired with an engineered prime editing guide RNA (pegRNA) that both directs
Cas9 to the
target genomic site and encodes the information for installing the desired
edit. Prime editing
proceeds through a multi-step editing process: 1) the Cas9 domain binds and
nicks the target
genomic DNA site, which is specified by the pegRNA's spacer sequence; 2) the
reverse
transcriptase domain uses the nicked genomic DNA as a primer to initiate the
synthesis of an
edited DNA strand using an engineered extension on the pegRNA as a template
for reverse
transcription¨this generates a single-stranded 3' flap containing the edited
DNA sequence; 3)
cellular DNA repair resolves the 3' flap intermediate by the displacement of a
5' flap species that
occurs via invasion by the edited 3' flap, excision of the 5' flap containing
the original DNA
sequence, and ligation of the new 3' flap to incorporate the edited DNA
strand, forming a
heteroduplex of one edited and one unedited strand; and 4) cellular DNA repair
replaces the
unedited strand within the heteroduplex using the edited strand as a template
for repair,
completing the editing process.
1161 Efficient incorporation of the desired edit requires that the newly
synthesized 3' flap
contains a portion of sequence that is homologous to the genomic DNA site.
This homology
enables the edited 3' flap to compete with the endogenous DNA strand (the
corresponding 5'
flap) for incorporation into the DNA duplex. Because the edited 3' flap will
contain less
sequence homology than the endogenous 5' flap, the competition is expected to
favor the 5' flap
strand. Thus, a potential limiting factor in the efficiency of prime editing
may be the failure of
the 3' flap, which contains the edit, to effectively invade and displace the
5' flap strand.
Moreover, successful 3' flap invasion and removal of the 5' flap only
incorporates the edit on
one strand of the double-stranded DNA genome. Permanent installation of the
edit requires
cellular DNA repair to replace the unedited complementary DNA strand using the
edited strand
as a template. While the cell can be made to favor replacement of the unedited
strand over the
edited strand (step 4 above) by the introduction of a nick in the unedited
strand adjacent to the
edit using a secondary sgRNA (i.e., the PE3 system), this process still relies
on a second stage of
DNA repair.
1171 This disclosure describes a modified approach to prime editing that
comprises
additionally inhibiting, blocking, or otherwise inactivating the DNA mismatch
repair (MMR)
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system. In certain embodiments, the DNA mismatch repair (MMR) system can be
inhibited,
blocked, or otherwise inactivating one or more proteins of the MMR system,
including, but not
limited to MLH1, PMS2 (or Mud, alpha), PMS1 (or MutL beta), MLH3 (or Mud.,
gamma),
MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01,
POLS, and PCNA. The disclosure contemplates any suitable means by which to
inhibit, block, or
otherwise inactivate the DNA mismatch repair (MMR) system, including, but not
limited to
inactivating one or more critical proteins of the MMR system at the genetic
level, e.g., by
introducing one or more mutations in the genes encoding a protein of the MMR
system, e.g.,
MLH1, PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS
alpha
(MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLO, and
PCNA.
[181 Thus, in one aspect, the present disclosure provides a method for editing
a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting,
evading, or otherwise
inactivating the DNA mismatch repair (MMR) system.
[191 In another aspect, the present disclosure provides a method for editing a
nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting,
evading, or otherwise
inactivating a protein of the MMR system, e.g., MUD, PMS2 (or MutL alpha),
PMS1 (or Mud,
beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3),
MSH2, MSH6, PCNA, RFC, EX01, POLO, and PCNA.
1201 In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting,
evading, or otherwise
inactivating MLH1 or variant thereof
1211 In another aspect, the present disclosure provides a method for editing a
nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting,
evading, or otherwise
inactivating PMS2 (or MutL alpha) or variant thereof.
1221 In yet another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting,
evading, or otherwise
inactivating PMS1 (or Mud, beta) or variant thereof
[231 in still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting,
evading, or otherwise
inactivating MLH3 (or MutL gamma) or variant thereof.
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1241 In another aspect, the present disclosure provides a method for editing a
nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting,
evading, or otherwise
inactivating MutS alpha (MSH2-MSH6) or variant thereof.
[251 In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting,
evading, or otherwise
inactivating MSH2 or variant thereof.
[261 In another aspect, the present disclosure provides a method for editing a
nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting,
evading, or otherwise
inactivating MSH6 or variant thereof.
1271 In yet another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting,
evading, or otherwise
inactivating PCNA or variant thereof.
[281 In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting,
evading, or otherwise
inactivating RFC or variant thereof.
[291 In another aspect, the present disclosure provides a method for editing a
nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting,
evading, or otherwise
inactivating EX01 or variant thereof.
1301 In yet another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting,
evading, or otherwise
inactivating POLS or variant thereof.
[311 Thus, in one aspect, the present disclosure provides a method for editing
a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting,
evading, or otherwise
inactivating the DNA mismatch repair (MMR) system.
1321 In another aspect, the disclosure provides a method for evading MMR by
installing one
or more silent mutations nearby an intended edit, resulting in the evading of
MMR and thereby
improving editing efficiency of prime editing. In various embodiments, the
number of silent
mutations installed can be one, or two, or three, or four, or five, or six, or
seven, or eight, or nine,
or ten, or eleven, or twelve, or thirteen, or fourteen, or fifteen, or
sixteen, or seventeen, or
eighteen, or nineteen, or twenty or more. The one more silent mutations may be
located
upstream or downstream (or a combination if multiple silent mutations are
involved) of the
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intended edit site, on the same or opposite strand of DNA as the intended edit
site (or a
combination if multiple silent mutations are involved). The silent mutations
may be located
upstream or downstream of the intended edit by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, or more
nucleotide positions away from the intended edit. In various embodiments, the
method of
evading by silent mutation installation results in a significant increase in
editing efficiency of
prime editing (e.g., at least 1.5-fold, at least 2.0-fold, at least 2.5-fold,
at least 3.0-fold, at least
3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least
5.5-fold, at least 6.0-fold, at
least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at
least 8.5-fold, at least 9.0-
fold, at least 9.5-fold, at least 10.0-fold, at least 11-fold, at least 12-
fold, at least 13-fold, at least
14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-
fold, at least 19-fold, at
least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least
24-fold, at least 25-
fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold,
at least 30-fold, at least
31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-
fold, at least 36-fold, at
least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least
41-fold, at least 42-
fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold,
at least 47-fold, at least
48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-
fold, at least 53-fold, at
least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least
58-fold, at least 59-
fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold,
at least 64-fold, at least
65-fold, at least 66-fold, at least 67-fold, at least 68-fold, at least 69-
fold, at least 70-fold, at least
71-fold, at least 72-fold, at least 73-fold, at least 74-fold, or at least 75-
fold increased) when one
or more silent mutations are installed nearby a desired site for installing a
genetic change by
prime editing, in the presence or absence of an inhibitor of MIVIR. In various
embodiments, the
method of evading MMR by silent mutation installation results in a significant
decrease in the
frequency of indel formation of prime editing (e.g., at least 1.5-fold, at
least 2.0-fold, at least 2.5-
fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-
fold, at least 5.0-fold, at least
5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least
7.5-fold, at least 8.0-fold, at
least 8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0-fold, at
least 11-fold, at least 12-
fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold,
at least 17-fold, at least
18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-
fold, at least 23-fold, at
least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least
28-fold, at least 29-
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fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold,
at least 34-fold, at least
35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-
fold, at least 40-fold, at
least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least
45-fold, at least 46-
fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold,
at least 51-fold, at least
52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-
fold, at least 57-fold, at
least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least
62-fold, at least 63-
fold, at least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold,
at least 68-fold, at least
69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at least 73-
fold, at least 74-fold, or at
least 75-fold decrease) when one or more silent mutations are installed nearby
a desired site for
installing a genetic change by prime editing, in the presence or absence of an
inhibitor of MMR.
[331 In another aspect, the present disclosure provides a method for editing a
nucleotide
molecule (e.g., a genome), comprising contacting a target nucleotide molecule
with a prime
editor and an inhibitor of the MMR system, e.g., an inhibitor of one or more
of MLH1, PMS2 (or
MutL alpha), PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS alpha (MSH2-
MSH6),
MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLS, or PCNA. In various
embodiments, the inhibitor can be a small molecule inhibitor. In other
embodiments, the
inhibitor can be an antibody, e.g., a neutralizing antibody. In still other
embodiments, the
inhibitor can be a variant of an MMR protein (e.g., a variant encoded by a
dominant negative
mutant of the gene encoding the MMR protein that adversely affects the
function or expression
of the normal wild type MMR protein, also referred to herein as a "dominant
negative mutant,"
"dominant negative variant," or a "dominant negative protein," e.g., a
"dominant negative MMR
protein"). In some embodiments, the inhibitor is a dominant negative variant
of an MMR protein
that inhibits the activity of a wild type MMR protein. For example, the
inhibitor can be an MLH1
protein variant (e.g., a dominant negative mutant) of one or more of IMLH1,
PMS2 (or MutL
alpha), PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6),
MutS beta
(MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLE, or PCNA, e.g., a dominant
negative
mutant of MLH1. In still other embodiments, the inhibitor can be targeted at
the level of
transcription, e.g., an siRNA or other nucleic acid agent that knocks down the
level of a
transcript encoding MLH1, IPMS2 (or 1MutL alpha), PMS1 (or Mutt. beta), IMLH3
(or MutL
gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC,
EX01, POLS, or PCNA. In yet other embodiments, the step of "contacting a
target nucleotide
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molecule with a prime editor" can include (i) delivering directly to a cell an
effective amount of
a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid
delivery system; (ii)
delivery to a cell an mRNA or delivery complex comprising an mRNA that encodes
a prime
editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g.,
an AAV or
lentivirus vector, plasmid, or other nucleic acid delivery vector) that
encodes a prime editor
fusion protein and/or a suitable pegRNA on one or more DNA vectors.
[341 In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating MLH1 or variant thereof. In another aspect, the present
disclosure provides a
method for editing a nucleotide molecule (e.g., a genome), comprising
contacting a target
nucleotide molecule with a prime editor and an inhibitor of MLH1. In various
embodiments, the
inhibitor can be a small molecule inhibitor. In other embodiments, the
inhibitor can be an anti-
MLH1 antibody, e.g., a neutralizing antibody that inactivates MLH1. In still
other embodiments,
the inhibitor can be a dominant negative mutant of MLH I. In still other
embodiments, the
inhibitor can be targeted at the level of transcription of MLH1, e.g., an
siRNA or other nucleic
acid agent that knocks down the level of a transcript encoding MLH1. In yet
other embodiments,
the step of "contacting a target nucleotide molecule with a prime editor" can
include (i)
delivering directly to a cell an effective amount of a prime editor fusion
protein (e.g., PEI or
PE2) complexed with a lipid delivery system; (ii) delivery to a cell an mRNA
or delivery
complex comprising an mRNA that encodes a prime editor fusion protein and/or a
suitable
pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or
other nucleic
acid delivery vector) that encodes a prime editor fusion protein and/or a
suitable pegRNA on one
or more DNA vectors.
[351 In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating PMS2 (or Mud- alpha) or variant thereof In another aspect, the
present disclosure
provides a method for editing a nucleotide molecule (e.g., a genome),
comprising contacting a
target nucleotide molecule with a prime editor and an inhibitor of PMS2 (or
MutL alpha). In
various embodiments, the inhibitor can be a small molecule inhibitor. In other
embodiments, the
inhibitor can be an anti-PMS2 (or MutL alpha) antibody, e.g., a neutralizing
antibody that
inactivates PMS2 (or MutL alpha). In still other embodiments, the inhibitor
can be a dominant
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negative mutant of PMS2 (or MutL alpha). In still other embodiments, the
inhibitor can be
targeted at the level of transcription of PMS2 (or MutL alpha), e.g., an siRNA
or other nucleic
acid agent that knocks down the level of a transcript encoding ML PMS2 (or
MutL alpha). In yet
other embodiments, the step of "contacting a target nucleotide molecule with a
prime editor" can
include (i) delivering directly to a cell an effective amount of a prime
editor fusion protein (e.g.,
PEI or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a
mRNA or delivery
complex comprising an mRNA that encodes a prime editor fusion protein and/or a
suitable
pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivina vector, plasmid, or
other nucleic
acid delivery vector) that encodes a prime editor fusion protein and/or a
suitable pegRNA on one
or more DNA vectors.
[361 In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating PM S1 (or MutL beta) or variant thereof. In another aspect, the
present disclosure
provides a method for editing a nucleotide molecule (e.g., a genome),
comprising contacting a
target nucleotide molecule with a prime editor and an inhibitor of PMS1 (or
MutL beta). in
various embodiments, the inhibitor can be a small molecule inhibitor. In other
embodiments, the
inhibitor can be an anti-PMS1 (or MutL beta) antibody, e.g., a neutralizing
antibody that
inactivates PMS1 (or MutL beta). In still other embodiments, the inhibitor can
be a dominant
negative mutant of PMS1 (or MutL beta). In still other embodiments, the
inhibitor can be
targeted at the level of transcription of PMS1 (or MutL beta), e.g., an siRNA
or other nucleic
acid agent that knocks down the level of a transcript encoding PMS1 (or MutL
beta). In yet other
embodiments, the step of "contacting a target nucleotide molecule with a prime
editor" can
include (i) delivering directly to a cell an effective amount of a prime
editor fusion protein (e.g.,
PEI or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a
mRNA or delivery
complex comprising an mRNA that encodes a prime editor fusion protein and/or a
suitable
pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivina vector, plasmid, or
other nucleic
acid delivery vector) that encodes a prime editor fusion protein and/or a
suitable pegRNA on one
or more DNA vectors.
[371 lEn still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating MLH3 (or MutL gamma) or variant thereof. In another aspect, the
present disclosure
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provides a method for editing a nucleotide molecule (e.g., a genome),
comprising contacting a
target nucleotide molecule with a prime editor and an inhibitor of MLH3 (or
MutL gamma). In
various embodiments, the inhibitor can be a small molecule inhibitor. In other
embodiments, the
inhibitor can be an anti-MLH3 (or MutL gamma) antibody, e.g., a neutralizing
antibody that
inactivates MLH3 (or MutL gamma). In still other embodiments, the inhibitor
can be a dominant
negative mutant of MLH3 (or MutL gamma). In still other embodiments, the
inhibitor can be
targeted at the level of transcription of P MLH3 (or MutL gamma), e.g., an
siRNA or other
nucleic acid agent that knocks down the level of a transcript encoding MLH3
(or MutL gamma).
In yet other embodiments, the step of "contacting a target nucleotide molecule
with a prime
editor" can include (i) delivering directly to a cell an effective amount of a
prime editor fusion
protein (e.g., PEI or PE2) complexed with a lipid delivery system; (ii)
delivery to a cell a mRNA
or delivery complex comprising an mRNA that encodes a prime editor fusion
protein and/or a
suitable peg,RNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector,
plasmid, or other
nucleic acid delivery vector) that encodes a prime editor fusion protein
and/or a suitable pegRNA
on one or more DNA vectors.
[381 In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating MutS alpha (MSH2-MSH6) or variant thereof. In another aspect, the
present
disclosure provides a method for editing a nucleotide molecule (e.g., a
genome), comprising
contacting a target nucleotide molecule with a prime editor and an inhibitor
of MutS alpha
(MSH2-MSH6). In various embodiments, the inhibitor can be a small molecule
inhibitor. In
other embodiments, the inhibitor can be an anti-MutS alpha (MSH2-MSH6)
antibody, e.g., a
neutralizing antibody that inactivates MutS alpha (MSH2-MSH6). In still other
embodiments,
the inhibitor can be a dominant negative mutant of MutS alpha (MSH2-MSH6). In
still other
embodiments, the inhibitor can be targeted at the level of transcription of
MutS alpha (MSH2-
MSH6), e.g., an siRNA or other nucleic acid agent that knocks down the level
of a transcript
encoding MutS alpha (MSH2-MSH6). In yet other embodiments, the step of
"contacting a target
nucleotide molecule with a prime editor" can include (i) delivering directly
to a cell an effective
amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a
lipid delivery
system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a
prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector
(e.g., an AAV or
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lentivirus vector, plasmid, or other nucleic acid delivery vector) that
encodes a prime editor
fusion protein and/or a suitable pegRNA on one or more DNA vectors.
1391 In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating MSH2 or variant thereof. In another aspect, the present
disclosure provides a method
for editing a nucleotide molecule (e.g., a genome), comprising contacting a
target nucleotide
molecule with a prime editor and an inhibitor of MSH2. In various embodiments,
the inhibitor
can be a small molecule inhibitor. In other embodiments, the inhibitor can be
an anti- MSH2
antibody, e.g., a neutralizing antibody that inactivates MSH2. In still other
embodiments, the
inhibitor can be a dominant negative mutant of MSH2. In still other
embodiments, the inhibitor
can be targeted at the level of transcription of MSH2, e.g., an siRNA or other
nucleic acid agent
that knocks down the level of a transcript encoding MSH2. In yet other
embodiments, the step of
"contacting a target nucleotide molecule with a prime editor" can include (1)
delivering directly
to a cell an effective amount of a prime editor fusion protein (e.g., PEI or
PE2) complexed with
a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex
comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii)
a DNA vector
(e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery
vector) that encodes a
prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
1401 In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating MSH6 or variant thereof. In another aspect, the present
disclosure provides a method
for editing a nucleotide molecule (e.g., a genome), comprising contacting a
target nucleotide
molecule with a prime editor and an inhibitor of MSH6. In various embodiments,
the inhibitor
can be a small molecule inhibitor. In other embodiments, the inhibitor can be
an anti-MSH6
antibody, e.g., a neutralizing antibody that inactivates MSH6. In still other
embodiments, the
inhibitor can be a dominant negative mutant of MSH6. In still other
embodiments, the inhibitor
can be targeted at the level of transcription of MSH6, e.g., an siRNA or other
nucleic acid agent
that knocks down the level of a transcript encoding MSH6. In yet other
embodiments, the step of
"contacting a target nucleotide molecule with a prime editor" can include (i)
delivering directly
to a cell an effective amount of a prime editor fusion protein (e.g., PEI. or
PE2) complexed with
a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex
comprising an mRNA
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that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii)
a DNA vector
(e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery
vector) that encodes a
prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
[411 In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating PCNA or variant thereof. In another aspect, the present
disclosure provides a method
for editing a nucleotide molecule (e.g., a genome), comprising contacting a
target nucleotide
molecule with a prime editor and an inhibitor of PCNA. In various embodiments,
the inhibitor
can be a small molecule inhibitor. In other embodiments, the inhibitor can be
an anti- PCNA
antibody, e.g., a neutralizing antibody that inactivates PCNA. In still other
embodiments, the
inhibitor can be a dominant negative mutant of PCNA. In still other
embodiments, the inhibitor
can be targeted at the level of transcription of PCNA, e.g., an siRNA or other
nucleic acid agent
that knocks down the level of a transcript encoding PCNA. In yet other
embodiments, the step of
"contacting a target nucleotide molecule with a prime editor" can include (i)
delivering directly
to a cell an effective amount of a prime editor fusion protein (e.g., PEI or
PE2) complexed with
a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex
comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii)
a DNA vector
(e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery
vector) that encodes a
prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
[421 In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating RFC or variant thereof. In another aspect, the present disclosure
provides a method
for editing a nucleotide molecule (e.g., a genome), comprising contacting a
target nucleotide
molecule with a prime editor and an inhibitor of RFC. In various embodiments,
the inhibitor can
be a small molecule inhibitor. In other embodiments, the inhibitor can be an
anti-RFC antibody,
e.g., a neutralizing antibody that inactivates RFC. In still other
embodiments, the inhibitor can be
a dominant negative mutant of RFC. In still other embodiments, the inhibitor
can be targeted at
the level of transcription of RFC, e.g., an siRNA or other nucleic acid agent
that knocks down
the level of a transcript encoding RFC. In yet other embodiments, the step of
"contacting a target
nucleotide molecule with a prime editor" can include (i) delivering directly
to a cell an effective
amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a
lipid delivery
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system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a
prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector
(e.g., an AAV or
lentivirus vector, plasmid, or other nucleic acid delivery vector) that
encodes a prime editor
fusion protein and/or a suitable pegRNA on one or more DNA vectors.
1431 In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating EX01 or variant thereof. In another aspect, the present
disclosure provides a method
for editing a nucleotide molecule (e.g., a genome), comprising contacting a
target nucleotide
molecule with a prime editor and an inhibitor of EX01. In various embodiments,
the inhibitor
can be a small molecule inhibitor. In other embodiments, the inhibitor can be
an anti-EX01
antibody, e.g., a neutralizing antibody that inactivates EX01. In still other
embodiments, the
inhibitor can be a dominant negative mutant of EX01. In still other
embodiments, the inhibitor
can be targeted at the level of transcription of EX01, e.g., an siRNA or other
nucleic acid agent
that knocks down the level of a transcript encoding EX01. In yet other
embodiments, the step of
"contacting a target nucleotide molecule with a prime editor" can include (i)
delivering directly
to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or
PE2) complexed with
a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex
comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii)
a DNA vector
(e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery
vector) that encodes a
prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
1441 In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating POLS or variant thereof In another aspect, the present disclosure
provides a method
for editing a nucleotide molecule (e.g., a genome), comprising contacting a
target nucleotide
molecule with a prime editor and an inhibitor of POLS. In various embodiments,
the inhibitor
can be a small molecule inhibitor. In other embodiments, the inhibitor can be
an anti-POLS
antibody, e.g., a neutralizing antibody that inactivates POLS. In still other
embodiments, the
inhibitor can be a dominant negative mutant of POLS. In still other
embodiments, the inhibitor
can be targeted at the level of transcription of POLO, e.g., an si RNA or
other nucleic acid agent
that knocks down the level of a transcript encoding POLO. In yet other
embodiments, the step of
"contacting a target nucleotide molecule with a prime editor" can include (i)
delivering directly
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to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or
PE2) complexed with
a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex
comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii)
a DNA vector
(e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery
vector) that encodes a
prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
[451 In one aspect, the present disclosure provides methods for editing a
nucleic acid molecule
by prime editing. In some embodiments, the method comprises contacting a
nucleic acid
molecule with a prime editor, a pegRNA, and an inhibitor of the DNA mismatch
repair pathway,
thereby installing one or more modifications to the nucleic acid molecule at a
target site.
[461 The method may increase the efficiency of prime editing and/or decrease
the frequency
of indel formation. In some embodiments, the prime editing efficiency is
increased by at least
1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least
3.5-fold, at least 4.0-fold, at
least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at
least 6.5-fold, at least 7.0-
fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-
fold, at least 9.5-fold, or at
least 10.0-fold in the presence of the inhibitor of the DNA mismatch repair
pathway. In some
embodiments, the frequency of indel formation is decreased by at least 1.5-
fold, at least 2.0-fold,
at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at
least 4.5-fold, at least 5.0-
fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-
fold, at least 7.5-fold, at least
8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least
10.0-fold in the presence
of the inhibitor of the DNA mismatch repair pathway.
[471 In some embodiments, the inhibitor of the DNA mismatch repair pathway
inhibits one or
more proteins of the DNA mismatch repair pathway. In some embodiments, the one
or more
proteins is selected from the group consisting of MLH1, PMS2 (or MutL alpha),
PMS1 (or MutL
beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MS116), MutS beta (MSH2-MSH3),
MSH2, MSH6, PCNA, RFC, EX01, POL8, and PCNA. In certain embodiments, the one
or more
proteins is MLH.1. In some embodiments, MLH1 comprises an amino acid sequence
of SEQ ID
NO: 204, or an amino acid sequence having at least 70%, at least 75%, at least
80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or up to
and including 100% sequence identity with SEQ ID NO: 204.
[481 The inhibitor utilized in the method may be an antibody, a small
molecule, a small
interfering RNA (siRNA), a small non-coding microRNA, or a dominant negative
variant of an
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MMR protein that inhibits the activity of a wild type MMR protein (e.g., a
dominant negative
variant of MLH1). In certain embodiments, the inhibitor is an antibody that
inhibits the activity
of one or more proteins of the DNA mismatch repair pathway. In some
embodiments, the
inhibitor is a small molecule that inhibits the activity of one or more
proteins of the DNA
mismatch repair pathway. In certain embodiments, the inhibitor is a small
interfering RNA
(siRNA) or a small non-coding microRNA that inhibits the activity of one or
more proteins of
the DNA mismatch repair pathway. In some embodiments, the inhibitor is a
dominant negative
variant of MLH1 that inhibits MLH1.
1491 In some embodiments, the dominant negative variant is (a) MLH1 E34A (SEQ
ID NO:
222), (b) MLH1 A756 (SEQ ID NO: 208), (c) MLH1 A754-756 (SEQ ID NO: 209), (d)
MLH1
E34A M54-756 (SEQ ID NO: 210), (e) MLH1 1-335 (SEQ ID NO: 211), (f) MLH1 1-335

E34A (SEQ ID NO: 212), (g) MLH1 1-335 NLSsv40 (SEQ ID NO: 213), (h) MLH1 501-
756
(SEQ ID NO: 215), (i) MLH I 501-753 (SEQ ID NO: 216), (j) MUD 461-753 (SEQ ID
NO:
218), or (k) NLSsv4 MLH1 501-753 (SEQ ID NO: 223), or a polypeptide
comprising an amino
acid sequence having at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and
including 100%
sequence identity with any of SEQ ID NOs: 208-213, 215, 216, 218, 222, or 223.
1501 The prime editors utilized in the methods of the present disclosure may
comprise multiple
components. In some embodiments, the prime editor comprises a napDNAbp and a
polymerase.
In some embodiments, the napDNAbp is a nuclease active Cas9 domain, a nuclease
inactive
Cas9 domain, or a Cas9 nickase domain or variant thereof. In certain
embodiments, the
napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d,
Cas12a, Cas12b1,
Cas13a, Cas12c, Cas12b2, Cas13a, Cas12c, Cas12d, Casi2e, Cas12h, Cas12i,
Cas12g, Cas12f
(Cas14), Casl2f1, Cas12j (Cas(Io), and Argonaute and optionally has a nickase
activity. In certain
embodiments, the napDNAbp comprises an amino acid sequence of any one of SEQ
ID NOs: 2,
4-67, or 99 (PEmax) or an amino acid sequence having at least an 80%, 85%,
90%, 95%, or 99%
sequence identity with any one of SEQ NOs: 2,4-67, or 99 (PEmax). In certain
embodiments,
the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 37
(e.g., the
napDNAbp of PEI and PE2) or an amino acid sequence having at least an 80%,
85%, 90%, 95%,
or 99% sequence identity with SEQ ID NO: 2. In some embodiments, the
polymerase is a DNA-
dependent DNA polymerase or an RNA-dependent DNA polymerase. In some
embodiments, the
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polymerase is a reverse transcriptase. In certain embodiments, the reverse
transcriptase
comprises an amino acid sequence of any one of SEQ ID NOs: 69-98 or an amino
acid sequence
having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one
of SEQ ID NOs:
69-98.
1511 The napDNAbp and the polymerase of the prime editor may be joined
together to form a
fusion protein. In some embodiments, the napDNAbp and the polymerase of the
prime editor are
joined by a linker to form a fusion protein. In certain embodiments, the
linker comprises an
amino acid sequence of any one of SEQ ID NOs: 102, or 118-131, or an amino
acid sequence
having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one
of SEQ ID NOs:
102, or 118-131. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 38, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
1521 The components used in the method (e.g., the prime editor, the pegRNA,
and/or the
inhibitor of the DNA mismatch repair pathway) may be encoded on a DNA vector.
In some
embodiments, the prime editor, the pegRNA, and the inhibitor of the DNA
mismatch repair
pathway are encoded on one or more DNA vectors. In certain embodiments, the
one or more
DNA vectors comprise AAV or lentivirus DNA vectors. In some embodiments, the
AAV vector
is serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
1531 The prime editors utilized in the presently disclosed methods may also be
further joined
to additional components. In some embodiments, the prime editor as a fusion
protein is further
joined by a second linker to the inhibitor of the DNA mismatch repair pathway.
In certain
embodiments, the second linker is a self-hydrolyzing linker. In certain
embodiments, the second
linker comprises an amino acid sequence of any one of SEQ ID NOs: 102, 118-
131, or 233-236,
or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99%
sequence identity
with any one of SEQ ID NOs: 102, 118-131, or 233-236. In some embodiments, the
second
linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45,
46,47, 48, 49, or 50
amino acids in length.
[541 lEn some embodiments, the one or more modifications to the nucleic acid
molecule
installed at the target site comprise one or more transitions, one or more
transversions, one or
more insertions, one or more deletions, or one more inversions. In certain
embodiments, the one
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or more transitions are selected from the group consisting of: (a) T to C; (b)
A to G; (c) C to T;
and (d) G to A. In certain embodiments, the one or more transversions are
selected from the
group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to
T; (f) A to C; (g) G to
C; and (h) G to T. In certain embodiments, the one or more modifications
comprises changing
(1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair,
(3) a G:C basepair to
a C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an
A:T basepair, (6) a
T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a
C:G basepair to a T:A
basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A
basepair, (11) an
A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair. In
some
embodiments, the one or more modifications comprises an insertion or deletion
of 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25
nucleotides.
[55] The methods of the present disclosure may be used for making corrections
to one or more
disease-associated genes. In some embodiments, the one or more modifications
comprises a
correction to a disease-associated gene. In certain embodiments, the disease-
associated gene is
associated with a polygenic disorder selected from the group consisting of:
heart disease; high
blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
In certain
embodiments, the disease-associated gene is associated with a monogenic
disorder selected from
the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1
Antitrypsin
Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia;
Hemochromatosis;
Huntington's Disease; Maple Syrup Urine Disease; Madan Syndrome;
Neurofibromatosis Type
1; Pachyonychia Congenita; Phenylketonuria; Severe Combined Immunodeficiency;
Sickle Cell
Disease; Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a prion
disease; and Tay-
Sachs Disease.
[561 In another aspect, the present disclosure provides compositions for
editing a nucleic acid
molecule by prime editing. In some embodiments, the composition comprises a
prime editor, a
pegRNA, and an inhibitor of the DNA mismatch repair pathway, wherein the
composition is
capable of installing one or more modifications to the nucleic acid molecule
at a target site.
[571 The composition may increase the efficiency of prime editing and/or
decrease the
frequency of indel formation. In some embodiments, the prime editing
efficiency is increased by
at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at
least 3.5-fold, at least 4.0-
fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-
fold, at least 6.5-fold, at least
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7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least
9.0-fold, at least 9.5-fold, or
at least 10.0-fold in the presence of the inhibitor of the DNA mismatch repair
pathway. In some
embodiments, the frequency of indel formation is decreased by at least 1.5-
fold, at least 2.0-fold,
at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at
least 4.5-fold, at least 5.0-
fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-
fold, at least 7.5-fold, at least
8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least
10.0-fold in the presence
of the inhibitor of the DNA mismatch repair pathway.
1581 In some embodiments, the inhibitor of the DNA mismatch repair pathway
inhibits one or
more proteins of the DNA mismatch repair pathway. In some embodiments, the one
or more
proteins is selected from the group consisting of MLH1, PMS2 (or MutL alpha),
PMS1 (or MutL
beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3),
MSH2, MSH6, PCNA, RFC, EX01, POLo, and PCNA. In certain embodiments, the one
or more
proteins is MLH1. In some embodiments, MLH1 comprises an amino acid sequence
of SEQ ID
NO: 204, or an amino acid sequence having at least 70%, at least 75%, at least
80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or up to
and including 100% sequence identity with SEQ ID NO: 204.
1591 The inhibitor utilized in the composition may be an antibody, a small
molecule, a small
interfering RNA (siRNA), a small non-coding microRNA, or a dominant negative
variant of an
MMR protein that inhibits the activity of a wild type MMR protein (e.g., a
dominant negative
variant of MLH1). In certain embodiments, the inhibitor is an antibody that
inhibits the activity
of one or more proteins of the DNA mismatch repair pathway. In some
embodiments, the
inhibitor is a small molecule that inhibits the activity of one or more
proteins of the DNA
mismatch repair pathway. In certain embodiments, the inhibitor is a small
interfering RNA
(siRNA) or a small non-coding microRNA that inhibits the activity of one or
more proteins of
the DNA mismatch repair pathway. In some embodiments, the inhibitor is a
dominant negative
variant of MLH1 that inhibits MLH1.
1601 In some embodiments, the dominant negative variant is (a) MLH1 E34A (SEQ
ID NO:
222), (b) MLH1 A756 (SEQ ID NO: 208), (c) MLH1 A754-756 (SEQ ID NO: 209), (d)
MLH1
E34A A754-756 (SEQ ID NO: 210), (e) MLLE 1-335 (SEQ ID NO: 211), (0 MLH1 1-335

E34A (SEQ ID NO: 212), (g) MLH1 1-335 NLSsys (SEQ ID NO: 213), (h) MLH1 501-
756
(SEQ ID NO: 215), (i)MLH1 501-753 (SEQ ID NO: 216), (j) MLH1 461-753 (SEQ ED
NO:
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218), or (k) NLSsv4 MUD 501-753 (SEQ ID NO: 223), or a polypeptide comprising
an amino
acid sequence having at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and
including 100%
sequence identity with any of SEQ ID NOs: 208-213, 215, 216, 218, 222, or 223.
1611 The prime editors utilized in the compositions of the present disclosure
comprise multiple
components. In some embodiments, the prime editor comprises a napDNAbp and a
polymerase.
In some embodiments, the napDNAbp is a nuclease active Cas9 domain, a nuclease
inactive
Cas9 domain, or a Cas9 nickase domain or variant thereof. In certain
embodiments, the
napDNAbp is selected from the group consisting of: Cas9, Cas12e, Cas12d,
Cas12a, Cas12b1,
Cas13a, Cas12c, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i,
Cas12g, Casl 2f
(Cas14), Casl2f1, Cas12j (Cas(I)), and Argonaute and optionally has a nickase
activity. In certain
embodiments, the napDNAbp comprises an amino acid sequence of any one of SEQ
ID NOs: 2,
4-67, or 99 (PEmax) or an amino acid sequence having at least an 80%, 85%,
90%, 95%, or 99%
sequence identity with any one of SEQ ID NOs: 2, 4-67, or 99 (PEmax). In
certain embodiments,
the napDNAbp comprises an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 37
(i.e., the
napDNAbp of PEI and PE2) or an amino acid sequence having at least an 80%,
85%, 90%, 95%,
or 99% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 37. In some
embodiments, the
polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA
polymerase. In
some embodiments, the polymerase is a reverse transcriptase. In certain
embodiments, the
reverse transcriptase comprises an amino acid sequence of any one of SEQ ID
NOs: 69-98 or an
amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence
identity with any
one of SEQ ID NOs: 69-98.
1621 The napDNAbp and the polymerase of the prime editor may be joined
together to form a
fusion protein. In some embodiments, the napDNAbp and the polymerase of the
prime editor are
joined by a linker to form a fusion protein. In certain embodiments, the
linker comprises an
amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or an amino acid
sequence
having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one
of SEQ ID NOs:
102, 118-131. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 38, 38, 39, 40,41,
42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
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[631 The components used in the compositions disclosed herein (e.g., the prime
editor, the
pegRNA, and/or the inhibitor of the DNA mismatch repair pathway) may be
encoded on a DNA
vector. In some embodiments, the prime editor, the pegRNA, and the inhibitor
of the DNA
mismatch repair pathway are encoded on one or more DNA vectors. In certain
embodiments, the
one or more DNA vectors comprise AAV or lentivirus DNA vectors. In some
embodiments, the
AAV vector is serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[641 The prime editors utilized in the presently disclosed compositions may
also be further
joined to additional components. In some embodiments, the prime editor as a
fusion protein is
further joined by a second linker to the inhibitor of the DNA mismatch repair
pathway. In certain
embodiments, the second linker is a self-hydrolyzing linker. In certain
embodiments, the second
linker comprises an amino acid sequence of any one of SEQ ID NOs: 102, 118-
131, or 233-236,
or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99%
sequence identity
with any one of SEQ ID NOs: 102, 118-131, or 233-236. In some embodiments, the
second
linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, or 50
amino acids in length.
[651 In some embodiments, the one or more modifications to the nucleic acid
molecule
installed at the target site comprise one or more transitions, one or more
transversions, one or
more insertions, one or more deletions, or one more inversions. In certain
embodiments, the one
or more transitions are selected from the group consisting of: (a) T to C; (b)
A to G; (c) C to T;
and (d) G to A. In certain embodiments, the one or more transversions are
selected from the
group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to
T; (f) A to C; (g) G to
C; and (h) G to T. In certain embodiments, the one or more modifications
comprises changing
(1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair,
(3) a G:C basepair to
a C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an
A:T basepair, (6) a
T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a
C:G basepair to a T:A
basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A
basepair, (11) an
A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair. In
some
embodiments, the one or more modifications comprises an insertion or deletion
of 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25
nucleotides.
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[661 The compositions of the present disclosure may be used for making
corrections to one or
more disease-associated genes. In some embodiments, the one or more
modifications comprises
a correction to a disease-associated gene. In certain embodiments, the disease-
associated gene is
associated with a polygenic disorder selected from the group consisting of:
heart disease; high
blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.
In certain
embodiments, the disease-associated gene is associated with a monogenic
disorder selected from
the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1
Antitrypsin
Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia;
Hemochromatosis;
Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome;
Neurofibromatosis Type
1; Pachyonychia Congenita; Phenylketonuria; Severe Combined Immunodeficiency;
Sickle Cell
Disease; Smith-Lemli-Opitz Syndrome; a trinucleotide repeat disorder; a prion
disease; and Tay-
Sachs Disease.
[671 In another aspect, this disclosure provides polynucleotides for editing a
DNA target site
by prime editing. In some embodiments, the polynucleotide comprises a nucleic
acid sequence
encoding a napDNAbp, a polymerase, and an inhibitor of the DNA mismatch repair
pathway,
wherein the napDNAbp and polymerase is capable in the presence of a pegRNA of
installing one
or more modifications in the DNA target site.
[681 The polynucleotide may increase the efficiency of prime editing and/or
decrease the
frequency of indel formation. In some embodiments, the prime editing
efficiency is increased by
at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at
least 3.5-fold, at least 4.0-
fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-
fold, at least 6.5-fold, at least
7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least
9.0-fold, at least 9.5-fold, or
at least 10.0-fold in the presence of the inhibitor of the DNA mismatch repair
pathway. In some
embodiments, the frequency of indel formation is decreased by at least 1.5-
fold, at least 2.0-fold,
at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at
least 4.5-fold, at least 5.0-
fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-
fold, at least 7.5-fold, at least
8.0-fold, at least 8.5-fold, at least 9.0-fold, at least 9.5-fold, or at least
10.0-fold in the presence
of the inhibitor of the DNA mismatch repair pathway.
[691 In some embodiments, the inhibitor of the DNA mismatch repair pathway
inhibits one or
more proteins of the DNA mismatch repair pathway. In some embodiments, the one
or more
proteins is selected from the group consisting of MLH1, PMS2 (or MutL alpha),
PMS1 (or MutL
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beta), MUD (or MutL gamma), MutS alpha (MSI12-MSH6), MutS beta (MSH2-MSH3),
MSH2, MSH6, PCNA, RFC, EX01, POL8, and PCNA. In certain embodiments, the one
or more
proteins is MLF11. In some embodiments, MLH1 comprises an amino acid sequence
of SEQ ID
NO: 204, or an amino acid sequence having at least 70%, at least 75%, at least
80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or up to
and including 100% sequence identity with SEQ ID NO: 204.
[701 The inhibitor utilized in the polynucleotide may be an antibody, a small
molecule, a small
interfering RNA (siRNA), a small non-coding microRNA, or a dominant negative
variant of an
MIVIR protein that inhibits the activity of a wild type MMR protein (e.g., a
dominant negative
variant of (MLLE). In certain embodiments, the inhibitor is an antibody that
inhibits the activity
of one or more proteins of the DNA mismatch repair pathway. In some
embodiments, the
inhibitor is a small molecule that inhibits the activity of one or more
proteins of the DNA
mismatch repair pathway. In certain embodiments, the inhibitor is a small
interfering RNA
(siRNA) or a small non-coding microRNA that inhibits the activity of one or
more proteins of
the DNA mismatch repair pathway. in some embodiments, the inhibitor is a
dominant negative
variant of MLH1 that inhibits MLH1.
1711 In some embodiments, the dominant negative variant is (a) MLH1 E34A (SEQ
ID NO:
222), (b) MLH1 A756 (SEQ ID NO: 208), (c) MLH1 M54-756 (SEQ ID NO: 209), (d)
MLH1
E34A A754-756 (SEQ ID NO: 210), (e) MLH1 1-335 (SEQ ID NO: 211), (f) MLH1 1-
335
E34A (SEQ ID NO: 212), (g) MLH1 1-335 NLSsv" (SEQ ID NO: 213), (h) MLH1 501-
756
(SEQ ID NO: 215), (i) MLH1 501-753 (SEQ ID NO: 216), (j) MLH1 461-753 (SEQ ID
NO:
218), or (k)NLSsm MLH1 501-753 (SEQ ID NO: 223), or a polypeptide comprising
an amino
acid sequence having at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to and
including 100%
sequence identity with any of SEQ ID NOs: 208-213, 215, 216, 218, 222, or 223.
1721 The prime editors utilized in the polynucleotides of the present
disclosure comprise
multiple components (e.g., a napDNAbp and a polymerase). In some embodiments,
the
napDNAbp is a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or
a Cas9
nickase domain or variant thereof. In certain embodiments, the napDNAbp is
selected from the
group consisting of: Cas9, Cas12e, Cas12d, Casi2a, Cas12b1, Cas13a, Cas12c,
Cas12b2,
Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14),
Casl2f1, Cas12j
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(Case), and Argonaute and optionally has a nickase activity. In certain
embodiments, the
napDNAbp comprises an amino acid sequence of any one of SEQ ID NOs: 2, 4-67,
or 99
(PEmax) or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or
99% sequence
identity with any one of SEQ ID NOs: 2,4-67, or 99 (PEmax). In certain
embodiments, the
napDNAbp comprises an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 37
(i.e., the
napDNAbp of PE1 and PE2) or an amino acid sequence having at least an 80%,
85%, 90%, 95%,
or 99% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 37. In some
embodiments, the
polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA
polymerase. In
some embodiments, the polymerase is a reverse transcriptase. In certain
embodiments, the
reverse transcriptase comprises an amino acid sequence of any one of SEQ TD
NOs: 69-98 or an
amino acid sequence having at least an 80%, 85%, 90%, 95%, or 99% sequence
identity with any
one of SEQ ID NOs: 69-98.
[731 The napDNAbp and the polymerase of the prime editor may be joined
together to form a
fusion protein. In some embodiments, the napDNAbp and the polymerase of the
prime editor are
joined by a linker to form a fusion protein. In certain embodiments, the
linker comprises an
amino acid sequence of any one of SEQ ID NOs: 102, 118-131, or an amino acid
sequence
having at least an 80%, 85%, 90%, 95%, or 99% sequence identity with any one
of SEQ ID NOs:
102, 118-131. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 38, 38, 39, 40,41,
42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
[741 The polynucleotides disclosed herein may comprise vectors. In some
embodiments, the
polynucleotide is a DNA vector. In certain embodiments, the DNA vector is an
AAV or
lentivirus DNA vector. In some embodiments, the AAV vector is serotype 1, 2,
3, 4, 5, 6, 7, 8, 9,
or 10.
[751 The prime editors encoded by the presently disclosed polynucleotides may
also be further
joined to additional components. In some embodiments, the prime editor as a
fusion protein is
further joined by a second linker to the inhibitor of the DNA mismatch repair
pathway. In certain
embodiments, the second linker comprises a self-hydrolyzing linker. In certain
embodiments, the
second linker comprises an amino acid sequence of any one of SEQ ID NOs: 102,
118-131, or
233-236, or an amino acid sequence having at least an 80%, 85%, 90%, 95%, or
99% sequence
identity with any one of SEQ ID NOs: 102, 118-131, or 233-236. In some
embodiments, the
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second linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, or
50 amino acids in length.
[761 In some embodiments, the one or more modifications to the nucleic acid
molecule
installed at the target site comprise one or more transitions, one or more
transversions, one or
more insertions, one or more deletions, or one more inversions. In certain
embodiments, the one
or more transitions are selected from the group consisting of: (a) T to C; (b)
A to G; (c) C to T;
and (d) G to A. In certain embodiments, the one or more transversions are
selected from the
group consisting of: (a) T to A; (b) T to G; (c) C to G; (d) C to A; (e) A to
T; (f) A to C; (g) G to
C; and (h) G to T. In certain embodiments, the one or more modifications
comprises changing
(1) a G:C basepair to a T:A basepair, (2) a G:C basepair to an A:T basepair,
(3) a G:C basepair to
a C:G basepair, (4) a T:A basepair to a G:C basepair, (5) a T:A basepair to an
A:T basepair, (6) a
T:A basepair to a C:G basepair, (7) a C:G basepair to a G:C basepair, (8) a
C:G basepair to a T:A
basepair, (9) a C:G basepair to an A:T basepair, (10) an A:T basepair to a T:A
basepair, (11) an
A:T basepair to a G:C basepair, or (12) an A:T basepair to a C:G basepair. In
some
embodiments, the one or more modifications comprises an insertion or deletion
of 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25
nucleotides.
1771 The polynucleotides of the present disclosure may be used for making
corrections to one
or more disease-associated genes. In some embodiments, the one or more
modifications
comprises a correction to a disease-associated gene. In certain embodiments,
the disease-
associated gene is associated with a polygenic disorder selected from the
group consisting of:
heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes;
cancer; and obesity.
In certain embodiments, the disease-associated gene is associated with a
monogenic disorder
selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency;
Alpha-1
Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy;
Galactosemia;
Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan
Syndrome;
Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylketonuria; Severe
Combined
Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; a
trinucleotide repeat
disorder; a prion disease; and Tay-Sachs Disease.
[781 In another aspect, the present disclosure provides cells. In some
embodiments, the cell
comprises any of the polynucleotides described herein.
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1791 In another aspect, the present disclosure provides pharmaceutical
compositions. In some
embodiments, the pharmaceutical composition comprises any of the compositions
disclosed
herein. In some embodiments, the pharmaceutical composition comprises any of
the
compositions disclosed herein and a pharmaceutically acceptable excipient. In
some
embodiments, the pharmaceutical composition comprises any of the
polynucleotides disclosed
herein. In some embodiments, the pharmaceutical composition comprises any of
the
polynucleotides disclosed herein and a pharmaceutically acceptable excipient.
1801 In another aspect, the present disclosure provides kits. In some
embodiments, the kit
comprises any of the compositions disclosed herein, a pharmaceutical
excipient, and instructions
for editing a DNA target site by prime editing. In some embodiments, the kit
comprises any of
the polynucleotides disclosed herein, a pharmaceutical excipient, and
instructions for editing a
DNA target site by prime editing.
1811 The present disclosure also provides methods and pegRNAs for prime
editing whereby
correction by the MMR pathway of the alterations introduced into a target
nucleic acid molecule
is evaded, without the need to provide an inhibitor of the MMR pathway.
Surprisingly, pegRNAs
designed with consecutive nucleotide mismatches compared to the endogenous
sequence of a
target site on a target nucleic acid, for example, pegRNAs that have three or
more consecutive
mismatching nucleotides, can evade correction by the MMR pathway, resulting in
an increase in
prime editing efficiency and a decrease in the frequency of indel formation
compared to the
introduction of a single nucleotide mismatch using prime editing. In addition,
insertions or
deletions of consecutive nucleotides at the target site of the target nucleic
acid, for example,
insertions or deletions greater than 10 nucleotides in length, introduced by
prime editing also
evade correction by the MMR pathway, resulting in an increase in prime editing
efficiency and a
decrease in the frequency of indel formation compared to the introduction of
an insertion or
deletion of less than 10 nucleotides in length using prime editing.
1821 Thus, in another aspect, the present disclosure provides methods for
editing a nucleic acid
molecule by prime editing comprising contacting a nucleic acid molecule with a
prime editor
(e.g., PE2, PE3, or any of the other prime editors described herein) and a
pegRNA with a DNA
synthesis template on its extension arm comprising three or more consecutive
nucleotide
mismatches relative to the endogenous sequence of a target site on the nucleic
acid molecule. In
some embodiments, at least one of the consecutive nucleotide mismatches
results in an alteration
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in the amino acid sequence of a protein expressed from the nucleic acid
molecule, while at least
one of the remaining nucleotide mismatches is a silent mutation. The silent
mutations may be in
coding regions of the target nucleic acid molecule (i.e., in a part of a gene
that encodes a
protein), or the silent mutations may be in non-coding regions of the target
nucleic acid
molecule. In some embodiments, when the silent mutations are in a coding
region, the silent
mutations introduce into the nucleic acid molecule one or more alternate
codons encoding the
same amino acid as the unedited nucleic acid molecule. In some embodiments,
when the silent
mutations are in a non-coding region, the silent mutations are present in a
region of the nucleic
acid molecule that does not influence splicing, gene regulation, RNA lifetime,
or other biological
properties of the target site on the nucleic acid molecule.
[831 Any number of consecutive nucleotide mismatches compared to the sequence
of the
target site can be designed in the DNA synthesis template of a pegRNA to
achieve the benefits of
evading correction by the IMMR pathway, and thereby increase prime editing
efficiency and/or
reduce indel formation. In some embodiments, the DNA synthesis template
comprises at least
three consecutive nucleotide mismatches compared to the sequence of the target
site. In some
embodiments, the DNA synthesis template of the extension arm on the pegRNA
comprises 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
or 25 consecutive
nucleotide mismatches relative to the endogenous sequence of a target site in
the nucleic acid
molecule edited by prime editing. In some embodiments, the DNA synthesis
template of the
extension arm on the pegRNA comprises three or more, four or more, five or
more, six or more,
seven or more, eight or more, nine or more, or ten or more consecutive
nucleotide mismatches
relative to the endogenous sequence of a target site on the nucleic acid
molecule. In certain
embodiments, the use of three or more consecutive nucleotide mismatches
results in an increase
in prime editing efficiency by at least 1.5-fold, at least 2.0-fold, at least
2.5-fold, at least 3.0-fold,
at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at
least 5.5-fold, at least 6.0-
fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-
fold, at least 8.5-fold, at least
9.0-fold, at least 9.5-fold, or at least 10.0-fold relative to a method using
a pegRNA comprising a
DNA synthesis template comprising only one consecutive nucleotide mismatch
relative to tbe
endogenous sequence of a target site on the nucleic acid molecule. In certain
embodiments, the
use of three or more consecutive nucleotide mismatches results in a decrease
in the frequency of
indel formation by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at
least 3.0-fold, at least
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3.5-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least
5.5-fold, at least 6.0-fold, at
least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at
least 8.5-fold, at least 9.0-
fold, at least 9.5-fold, or at least 10.0-fold relative to a method using a
pegRNA comprising a
DNA synthesis template comprising only one consecutive nucleotide mismatch
relative to the
endogenous sequence of a target site on the nucleic acid molecule.
[841 In another aspect, the present disclosure provides methods for editing a
nucleic acid
molecule by prime editing comprising contacting a nucleic acid molecule with a
prime editor
(e.g., PE2, PE3, or any of the other prime editors described herein) and a
pegRNA with a DNA
synthesis template on its extension arm comprising an insertion or deletion of
10 or more
contiguous nucleotides relative to the endogenous sequence of a target site on
the nucleic acid
molecule. In some embodiments, the DNA synthesis template of a pegRNA can be
designed to
introduce insertions or deletions greater than 3 nucleotides to avoid or
reduce the impact of
mismatch correction by the cellular MMR. pathway, thereby improving prime
editing efficiency.
In some embodiments, the DNA synthesis template of the pegRNA is designed to
introduce one
or more insertions and/or deletions of 3, 4, 5, 6, 7, 8, 9, 10, or more
contiguous nucleotides to
avoid or reduce the impact of mismatch correction by the cellular MlvIR
pathway, thereby
improving prime editing efficiency. In some embodiments, insertions or
deletions of any length
greater than 10 contiguous nucleotides can be used to achieve the benefits of
evading correction
by the MMR pathway. In some embodiments, the DNA synthesis template comprises
an
insertion of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20,
21, 22, 23, 24, or 25
contiguous nucleotides relative to the endogenous sequence of a target site on
a nucleic acid
molecule edited by prime editing. In some embodiments, the DNA synthesis
template comprises
a deletion of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, or 25
contiguous nucleotides relative to the endogenous sequence of a target site on
a nucleic acid
molecule edited by prime editing. In some embodiments, the DNA synthesis
template comprises
an insertion of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides relative to the
endogenous sequence
of a target site on a nucleic acid molecule edited by prime editing. In some
embodiments, the
DNA synthesis template comprises a deletion of 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40
contiguous nucleotides
relative to the endogenous sequence of a target site on a nucleic acid
molecule edited by prime
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editing. In some embodiments, the DNA synthesis template comprises an
insertion or deletion of
11 or more contiguous nucleotides, 12 or more contiguous nucleotides, 13 or
more contiguous
nucleotides, 14 or more contiguous nucleotides, 15 or more contiguous
nucleotides, 16 or more
contiguous nucleotides, 17 or more contiguous nucleotides, 18 or more
contiguous nucleotides,
19 or more contiguous nucleotides, 20 or more contiguous nucleotides, 21 or
more contiguous
nucleotides, 22 or more contiguous nucleotides, 23 or more contiguous
nucleotides, 24 or more
contiguous nucleotides, or 25 or more contiguous nucleotides relative to a
target site on a nucleic
acid molecule. In certain embodiments, the DNA synthesis template comprises an
insertion or
deletion of 15 or more contiguous nucleotides relative to the endogenous
sequence of a target
site on the nucleic acid molecule.
[851 In some embodiments, prime editing with a pegRNA designed to introduce an
insertion
and/or deletion of multiple contiguous nucleotides, for example, three or more
contiguous
nucleotides, relative to the endogenous sequence of a target site results in
an increase in prime
editing efficiency compared to prime editing with a corresponding control
pegRNA (e.g., a
control pegRNA that does not introduce an insertion or deletion of three or
more contiguous
nucleotides) by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at
least 3.0-fold, at least 3.5-
fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-
fold, at least 6.0-fold, at least
6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least
8.5-fold, at least 9.0-fold, at
least 9.5-fold, or at least 10.0-fold. In some embodiments, prime editing with
a pegRNA
designed to introduce an insertion or deletion of 3, 4, 5, 6, 7, 8, 9, 10, or
more contiguous
nucleotides relative to the endogenous sequence of a target site results in an
increase in prime
editing efficiency relative to prime editing with a corresponding control
pegRNA (e.g., a control
pegRNA that does not introduce insertion or deletion of the three or more
contiguous
nucleotides) by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at
least 3.0-fold, at least 3.5-
fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-
fold, at least 6.0-fold, at least
6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least
8.5-fold, at least 9.0-fold, at
least 9.5-fold, or at least 10.0-fold. In some embodiments, making an
insertion or deletion of 10
or more contiguous nucleotides results in an increase in prime editing
efficiency by at least 1.5-
fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-
fold, at least 4.0-fold, at least
4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least
6.5-fold, at least 7.0-fold, at
least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at
least 9.5-fold, or at least 10.0-
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fold relative to a method using a pegRNA comprising a DNA synthesis template
comprising an
insertion or deletion of fewer than 10 nucleotides relative to the endogenous
sequence of a target
site on the nucleic acid molecule. In some embodiments, making an insertion or
deletion of 10 or
more nucleotides results in a decrease in the frequency of indel formation by
at least 1.5-fold, at
least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at
least 4.0-fold, at least 4.5-
fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-
fold, at least 7.0-fold, at least
7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least
9.5-fold, or at least 10.0-fold
relative to a method using a pegRNA comprising a DNA synthesis template
comprising an
insertion or deletion of fewer than 10 nucleotides relative to the endogenous
sequence of a target
site on the nucleic acid molecule.
[861 In another aspect, the present disclosure also provides pegRNAs useful
for editing a
nucleic acid molecule by prime editing while evading correction by the MMR
pathway of the
alterations introduced into the nucleic acid molecule, thereby increasing
prime editing efficiency
and/or reducing indel formation. In some embodiments, the extension arm of the
pegRNAs
provided by the present disclosure comprise three or more consecutive
nucleotide mismatches
relative to the endogenous sequence of a target site on the nucleic acid
molecule. In some
embodiments, at least one of the three consecutive nucleotide mismatches
relative to the
endogenous sequence of the target site is a silent mutation. In some
embodiments, at least one of
the consecutive nucleotide mismatches results in an alteration in the amino
acid sequence of a
protein expressed from the target nucleic acid molecule, while at least one of
the remaining
nucleotide mismatches is a silent mutation. The silent mutations may be in
coding regions of the
target nucleic acid molecule (i.e. ,in a part of a gene that encodes a
protein), or the silent
mutations may be in non-coding regions of the target nucleic acid molecule. In
some
embodiments, when the silent mutations are in a coding region, the silent
mutations introduce
into the nucleic acid molecule one or more alternate codons encoding the same
amino acid as the
unedited nucleic acid molecule. In some embodiments, when the silent mutations
are in a non-
coding region, the silent mutations are present in a region of the nucleic
acid molecule that does
not influence splicing, gene regulation, RNA lifetime, or other biological
properties of the target
site on the nucleic acid molecule.
[871 Any number of consecutive nucleotide mismatches of three or more can be
incorporated
into the extension arm of the pegRNAs described herein to achieve the benefits
of evading
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correction by the MMR pathway. In some embodiments, the DNA synthesis template
of the
extension arm of the pegRNA comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, or 25 consecutive nucleotide mismatches relative to
the endogenous
sequence of a target site on the nucleic acid molecule edited by prime
editing. In some
embodiments, the DNA synthesis template of the extension arm of the pegRNA
comprises at
least three consecutive nucleotide mismatches relative to the endogenous
sequence of a target
site on the nucleic acid molecule edited by prime editing. In some
embodiments, the DNA
synthesis template of the extension arm of the pegRNA comprises 3, 4, 5, 6, 7,
8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotide mismatches relative
to the endogenous
sequence of a target site on the nucleic acid molecule edited by prime
editing. In some
embodiments, the DNA synthesis template of the extension arm of the pegRNA
comprises 3, 4,
5, 6, 7, 8, 9, or 10 consecutive nucleotide mismatches relative to the
endogenous sequence of a
target site on the nucleic acid molecule edited by prime editing. In some
embodiments, the DNA
synthesis template of the extension arm on the pegRNA comprises three or more,
four or more,
five or more, six or more, seven or more, eight or more, nine or more, or ten
or more consecutive
nucleotide mismatches relative to the endogenous sequence of a target site on
the nucleic acid
molecule. In certain embodiments, the presence of three or more consecutive
nucleotide
mismatches on the extension arm of the pegRNA results in an increase in prime
editing
efficiency by at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at
least 3.0-fold, at least 3.5-fold,
at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at
least 6.0-fold, at least 6.5-
fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-
fold, at least 9.0-fold, at least
9.5-fold, or at least 10.0-fold relative to a pegRNA comprising a DNA
synthesis template
comprising only one consecutive nucleotide mismatch relative to the endogenous
sequence of a
target site on the nucleic acid molecule. In certain embodiments, the use of
three or more
consecutive nucleotide mismatches results in a decrease in the frequency of
indel formation by at
least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at
least 3.5-fold, at least 4.0-
fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-
fold, at least 6.5-fold, at least
7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least
9.0-fold, at least 9.5-fold, or
at least 10.0-fold relative to a pegRNA comprising a DNA synthesis template
comprising only
one consecutive nucleotide mismatch relative to the endogenous sequence of a
target site on the
nucleic acid molecule.
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[881 In another aspect, the present disclosure provides a prime editor system
for site specific
genome modification comprising (a) a prime editor comprising (i) a nucleic
acid programmable
DNA binding protein (napDNAbp) and (ii) a DNA polymerase, and (b) an inhibitor
of the DNA
mismatch repair pathway. In some embodiments, the inhibitor of the DNA
mismatch repair
pathway inhibits one or more proteins of the DNA mismatch repair pathway
(e.g., MLH1, PMS2
(or MutL alpha), PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS alpha (MSH2-
MSH6),
MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLE, and/or PCNA). In
some
embodiments, the one or more proteins is MLH1. In certain embodiments, the
MLH1 comprises
an amino acid sequence of SEQ ID NO: 204, or an amino acid sequence having at
least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least
96%, at least 97%, at
least 98%, at least 99%, or up to and including 100% sequence identity with
SEQ ID NO: 204.
[891 Any inhibitor of the DNA mismatch repair pathway may be used in the
systems described
herein. In some embodiments, the inhibitor is an antibody that inhibits the
activity of one or more
proteins of the DNA mismatch repair pathway. In some embodiments, the
inhibitor is a small
molecule that inhibits the activity of one or more proteins of the DNA
mismatch repair pathway.
In some embodiments, the inhibitor is a small interfering RNA (siRNA) or a
small non-coding
microRNA that inhibits the activity of one or more proteins of the DNA
mismatch repair
pathway. In some embodiments, the inhibitor is a dominant negative variant of
an MMR protein
that inhibits the activity of a wild type MMR protein (e.g., a dominant
negative variant of MLH1
that inhibits MLH1).
[901 In certain embodiments, the dominant negative variant used in the systems
of the present
disclosure is (a) MLH1 E34A (SEQ ID NO: 222), (b) MLH1 A756 (SEQ ID NO: 208),
(c)
MLH1 A754-756 (SEQ ID NO: 209), (d) MLH1 E34A A754-756 (SEQ ID NO: 210), (e)
MLH1
1-335 (SEQ ID NO: 211), (f) MLH1 1-335 E34A (SEQ ED NO: 212), (g) MLH1 1-335
NLSSV40 (SEQ ID NO: 213), (h) MLH1 501-756 (SEQ ID NO: 215), (i) MLH1 501-753
(SEQ
ID NO: 216), (j) MLH1 461-753 (SEQ ID NO: 218), or (k) NLSSV40 MLIT1 501-753
(SEQ ID
NO: 223), or a polypeptide comprising an amino acid sequence having at least
70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at
least 97%, at least
98%, at least 99%, or up to and including 100% sequence identity with any of
SEQ ID .N0s:
208-213, 215, 216, 218, 222, or 223. The present disclosure also contemplates
methods for
performing prime editing on a nucleic acid molecule in a cell in which MMR
activity is knocked
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out entirely (e.g., by knocking down one or more genes involved in the MMR
pathway in the
genome of the cell). Such methods provide the benefits of inhibiting MMR
(e.g., improved
editing efficiency and decreased indel formation) without the need to provide
an inhibitor of
MMR. Thus, in another aspect, the present disclosure provides methods for
editing a nucleic
acid molecule by prime editing comprising: contacting a nucleic acid molecule
with a prime
editor and a pegRNA, thereby installing one or more modifications to the
nucleic acid molecule
at a target site, wherein the nucleic acid molecule is in a cell comprising a
knockout of one or
more genes involved in the DNA mismatch repair (MMR) pathway. In some
embodiments, the
method further comprises contacting the nucleic acid molecule with a second
strand nicking
gRNA. In certain embodiments, the prime editing efficiency is increased by at
least 1.5-fold, at
least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at
least 4.0-fold, at least 4.5-
fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-
fold, at least 7.0-fold, at least
7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least
9.5-fold, or at least 10.0-fold
relative to a method performed in a cell that does not comprise a knockout of
one or more genes
involved in MMR. In certain embodiments, the frequency of indel formation is
decreased by at
least I.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at
least 3.5-fold, at least 4.0-
fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-
fold, at least 6.5-fold, at least
7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 8.5-fold, at least
9.0-fold, at least 9.5-fold, or
at least 10.0-fold relative to a method performed in a cell that does not
comprise a knockout of
one or more genes involved in MMR. In some embodiments, the one or more genes
involved in
MMR is selected from the group consisting of genes encoding the proteins MLH1,
PMS2 (or
MutL alpha), PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS alpha (MSH2-
MSH6),
MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POL6, and PCNA. In certain

embodiments, the one or more genes is the gene encoding MLH1 (e.g., comprising
an amino
acid sequence of SEQ ID NO: 204, or an amino acid sequence having at least
70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at
least 99%, or up to and including 100% sequence identity with SEQ ID NO: 204).
[911 In another aspect, the present disclosure provides methods for editing a
nucleic acid
molecule by prime editing comprising: contacting a nucleic acid molecule with
a prime editor, a
pegRNA, and an inhibitor of p53, thereby installing one or more modifications
to the nucleic
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acid molecule at a target site. In some embodiments, the method further
comprises contacting the
nucleic acid molecule with a second strand nicking gRNA.
1921 In some embodiments, the prime editing efficiency is increased by at
least 1.5-fold, at
least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at
least 4.0-fold, at least 4.5-
fold, at least 5.0-fold, at least 5.5-fold, at least 6.0-fold, at least 6.5-
fold, at least 7.0-fold, at least
7.5-fold, at least 8.0-fold, at least 8.5-fold, at least 9.0-fold, at least
9.5-fold, at least 10.0-fold, at
least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least
15-fold, at least 16-fold,
at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at
least 21-fold, at least 22-
fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold,
at least 27-fold, at least
28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-
fold, at least 33-fold, at
least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least
38-fold, at least 39-
fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold,
at least 44-fold, at least
45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-
fold, at least 50-fold, at
least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least
55-fold, at least 56-fold,
at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at
least 61-fold, at least 62-
fold, at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold,
at least 67-fold, at least
68-fold, at least 69-fold, at least 70-fold, at least 71-fold, at least 72-
fold, at least 73-fold, at least
74-fold, or at least 75-fold, in the presence of the inhibitor of p53. In some
embodiments, the
frequency of indel formation is decreased by at least 1.5-fold, at least 2.0-
fold, at least 2.5-fold,
at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 4.5-fold, at
least 5.0-fold, at least 5.5-
fold, at least 6.0-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-
fold, at least 8.0-fold, at least
8.5-fold, at least 9.0-fold, at least 9.5-fold, at least 10.0-fold, at least
11-fold, at least 12-fold, at
least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least
17-fold, at least 18-
fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold,
at least 23-fold, at least
24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-
fold, at least 29-fold, at
least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least
34-fold, at least 35-
fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold,
at least 40-fold, at least
41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-
fold, at least 46-fold, at
least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least
51-fold, at least 52-
fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold,
at least 57-fold, at least
58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-
fold, at least 63-fold, at
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least 64-fold, at least 65-fold, at least 66-fold, at least 67-fold, at least
68-fold, at least 69-fold,
at least 70-fold, at least 71-fold, at least 72-fold, at least 73-fold, at
least 74-fold, or at least 75-
fold, in the presence of the inhibitor of p53.
1931 In some embodiments, the inhibitor of p53 is a protein. In certain
embodiments, the
inhibitor of p53 is the protein 153. In some embodiments, the inhibitor of p53
is an antibody that
inhibits the activity of p53. In some embodiments, the inhibitor of p53 is a
small molecule that
inhibits the activity of p53. In some embodiments, the inhibitor of p53 is a
small interfering
RNA (siRNA) or a small non-coding microRNA that inhibits the activity of p53.
194I In another aspect, the present disclosure describes improved prime editor
fusion proteins,
including PEmax of SEQ ID NO: 99. The disclosure also contemplates fusion
proteins having
an amino acid sequence with a sequence identity of at least 80%, at least 85%,
at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least
up to 100% with SEQ
ID NO: 99.
1951 The inventors have surprisingly found that the editing efficiency of
prime editing may be
significantly increased (e.g., 2-fold increase, 3-fold increase, 4-fold
increase, 5-fold increase, 6-
fold increase, 7-fold increase, 8-fold increase, 9-fold increase, or 10-fold
increase or more) when
one or more components of the canonical prime editor fusion protein (i.e.,
PE2) are modified.
Modifications may include a modified amino acid sequence of one or more
components (e.g., a
Cas9 component, a reverse transcriptase component, or a linker).
[961 In other aspects, the present disclosure also provides compositions and
pharmaceutical
compositions comprising PEmax, methods of prime editing using PEmax,
polynucleotides and
vectors encoding PEmax, and kits and cells comprising PEmax.
[971 It should be appreciated that the foregoing concepts, and additional
concepts discussed
below, may be arranged in any suitable combination, as the present disclosure
is not limited in
this respect. Further, other advantages and novel features of the present
disclosure will become
apparent from the following detailed description of various non-limiting
embodiments when
considered in conjunction with the accompanying figures.
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BRIEF DESCRIPTION OF THE DRAWINGS
[98] The following drawings form part of the present specification and are
included to further
demonstrate certain aspects of the present disclosure, which can be better
understood by
reference to one or more of these drawings in combination with the detailed
description of
specific embodiments presented herein.
[991 FIG. 1 provides a schematic showing that prime editing enables guide RNA-
templated
genomic manipulations. DNA prime editing intermediates capable of being
repaired by cellular
factors are shown in boxes.
[100] FIG. 2 provides a schematic for a DNA repair CRISPRi screen for prime
editing
outcomes.
11011 FIGs. 3A-3C show optimization of prime editing efficiency at the target
site. FIG. 3A
provides a schematic for the optimization process. FIG. 3B shows percent reads
with a specified
modification at a target site in HeLa cells. FIG. 3C shows percent reads with
a specified
modification at a target site with blasticidin selection in HeLa cells.
[102] IFIGs. 4A-4B show a prime editing CRISPRi screen with a DNA repair
library. FIG. 4A
provides a schematic of the screening process. FIG. 4B shows percent reads
with a specified
modification in bulk editing of post-screen HeLa cells.
[103] FIGs. 5A-5B show that the CRISPRi screen reveals that DNA mismatch
repair limits
prime editing efficiency. Knockdown of mismatch repair proteins (MSH2, MS116,
PMS2, and
MLH1) improves the efficiency of PE2 by 3-fold and PE3 by 2-fold.
[104] FIGS. 6A-6C show that siRNA knockdown of MMR improves prime editing in
HEK293T cells. Editing results at multiple endogenous loci validate the
findings of the CRISPRi
screen.
[105] :FIGS. 7A-7B show that complete MMR knockout dramatically enhances prime
editing.
In the absence of MMR, PE2 editing efficiency is shown to match PE3 editing
efficiency.
[106] FIG. 8A provides a schematic for the mechanism of mismatch repair (MMR).
In the first
step, MSH2:MSH6 (MutSa) binds the mismatch and recruits MLH1:PMS2 (MutLa). The
DNA
nick signals to MMR which strand to repair. In the second step, MutLa
indiscriminately incises
the nicked strand 5' and 3' of the mismatch. In the third step, EX01 excises
the mismatch from
MutLa-generated nicks. In the fourth step, POLE resynthesizes the excised
strand, followed by
LIG1 ligation.
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[107] FIG. 8B provides yet another schematic for the mechanism of mismatch
repair, MMR., in
eukaryotic cells. The left side of the schematic depicts 5' MMR. (A) The MutS
homolog proteins
(MSH, purple) MutSa (MSH2-MSH6), or MutSI3 (MSH2-MSH3) recognize and bind a
mismatch. RPA bound to single-strand DNA prevents EX01 from accessing and
degrading
DNA. (B) In the sliding clamp model, MutSa/r3 at a mismatch binds ATP and
undergoes
nucleotide switch activation, becoming a sliding clamp that diffuses along the
DNA. Multiple
MSH clamps are loaded at a single mismatch. The interaction of EX01 with MSH
sliding
clamps overcomes the RPA bather and activates EX01 for 5' to 3' excision from
the 5' nick.
MutL homolog proteins (MLH) (MutLa is ScM1h1-Pms1 or HsMLH1-PMS2) bind ATP and

may interact with MSH sliding clamps, though MLH is not absolutely required in
vitro for 5'
MMR. In other models, MSH remains at the mismatch to authorize excision or can
load multiple
MLH clamps onto the DNA in the vicinity of the mismatch (not shown). (C) In
the sliding clamp
model, the EXOi/MSHI complex dissociates after excising several hundred
nucleotides. Iterative
rounds of MSH-EX01 excision create an excision tract coated with RPA that
extends from the 5'
nick to just beyond the mismatch. MLH may limit excision by modulating the
number of MSH
clamps on DNA. (D) RFC (not shown) loads PCNA clamps with specific orientation
at 3' termini
of strand breaks or gaps, and PCNA facilitates high-fidelity DNA synthesis by
Pol 8 or e. (E)
DNA ligase I seals the nick. The right side of the schematic depicts 3' MMR.
(A) MSH
recognizes a mismatch. (B) In the sliding clamp model, ATP-dependent binding
and nucleotide
switching creates MSH sliding clamps that diffuse from the mismatch. The
interaction of ATP-
bound MLH heterodimers with MSH sliding clamps and PCNA oriented with respect
to 3'
termini activates MLH strand-specific nicking. Alternatively, ATP-activated
MSH may remain at
the mismatch to load MLH and activate nicking (not shown). (C) Excision is
EX01-dependent
or -independent, leading to an RPA-coated excision track. An EX01-independent
IPol 8 strand-
displacement pathway is not shown. (D) Pol 6 or a with the aid of PCNA
completes gap filling.
(E) DNA ligase I seals the nick.
[108] FIGs. 9A-9C provide a schematic of mismatch repair of PE2 intermediates.
MMR
inhibition provides additional time for flap ligation, removing the strand
discrimination signal for
repair of the heteroduplex.
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[109] FIG. 10 shows that expression of dominant-negative MLH1 mutants boosts
PE2
efficiency. MLH1 dominant-negative mutants improve PE2 efficiency by 2- to 4-
fold. RNF2 +3
G to C is not responsive to MMR-inhibition.
[110] FIGs. 11A-118 show the effect of MUil mutants on PE3. MLH1 mutants
reduce PE3
indels by half.
[111] IFIGs. 12A-12B show that MLH1 mutant improvements translate to other
sites. FIG. 12A
shows that PE2 editing efficiency increases with MLH1 mutants, and only RNF2
+3 G to C is
resistant to MMR-inhibition. FIG. 12B shows that MLH1 mutants reduce the
occurrence of
indels by half.
[112] FIG. 13 provides a schematic showing mismatch repair of PE3
intermediates.
[113] FIG. 14 provides a schematic showing that mismatch repair differentially
resolves PE3
intermediates. Mismatch repair is required for the one edit-favored
intermediate.
[114] FIG. 15A-1511 show screening of MLH1 mutants for smaller size and
improved activity.
FIG. 15A shows that MLH1 A754-756 most strongly promotes PE2 editing
(hereafter named
MLHldn). MLH1 N-terminal domain approaches the effectiveness of MU-Ildn
(hereafter named
MLHldn'TD). MLH1 dominant negative mutants may function by saturating binding
of MutS.
FIG. 15B shows that the MLH1 N-terminal domain + NLS approaches the activity
of MLH1neg.
FIG. 15C shows that MLHldn fusion to PE by a self-cleavable P2A linker (PE-2A-
MLH1dn)
can improve prime editing efficiency. FIGs. 15D-15F show that MMR KD
phenocopies
MLH1neg expression. FIGs. 15G-151I show that the efficiency of PE2 and PE3 is
equal in the
absence of MMR, suggesting that the complementary nick only serves to bias
MMR.
[115] FIG. 16 shows that MLHidn reduces indels for PE3. Silent pegRNA is
pegRNA that
does not encode an edit or produce a mismatch. MLHldn only reduces PE3 indels
if a mismatch
is generated.
[116] FIG. 17 show that mismatch repair of PE heteroduplexes produces a
diffuse indel
pattern. Indel distribution is broad for PE3 for these edits, but inhibiting
MMR with IVILHldn
narrows that distribution. This suggests that MMR makes incisions after
mismatch recognition
that contribute to the indels generated by PE3.
[117] FIG. 18 shows mismatch repair of PE3 intermediates.
[118] FIGs. 19A-19B show that MMR excision of the target locus generates
indels in PE3.
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[119] FIGs. 20A-20B show that MMR knockdown or knockout has no effect on RNF2 -
F3 G to
C. This suggests that the RNF2 site is not repaired by MMR or the resulting
C:C mismatch is not
repaired by MMR.
[120] FIGs. 21A-21C show that other substitution edits at RNF2 can be improved
with
MLHldn.
[121] FIGs. 22A-22B show that MLHldn improves substitution edits at other
sites, including
HEK3. MLHidn strongly enhances PE2 editing and lowers PE3 indels.
[122] FIGs. 23A-23D show that MLHldn improves substitution edits at other
sites, including
FANCF. MLHldn strongly enhances PE2 editing and lowers PE3 indels.
[123] FIGs. 24A-24B show that PE improvement by MHLldn is mismatch dependent.
MLHIdn increases PE2 editing by 2-fold on average in HEK293T cells. FIG. 24A
shows that G
to C edits (C:C mismatches) are unaffected by MMR in HEK293T cells. This
suggests that G to
C edits have a higher baseline efficiency than other substitutions. FIG. 24B
shows a substantial
increase in the ratio of edit:indel purity from MLH ldn used with PE3, which
is also mismatch
dependent.
[1241 FIGs. 25A-25D show that MLHldn also improves the efficiency of small
insertion and
deletion edits. MMR is known to repair insertions and deletions <15
nucleotides in length.
[125] FIGs. 26A-26B show that MLHldn reduced pegRNA scaffold integration.
Scaffold
integration events at these sites occur through a double-strand break (DSB)
intermediate.
[126] FIG. 27 shows that MLHidn does not promote substantial PE off-target
editing. Small
increases in off-target (OT) editing were observed at the HEK4 off-target site
3.
[127] FIGs. 28A-28B show that MLHldn does not induce detectable microsatellite
instability
at biomarker loci. MMR inhibition is known to cause shortening of homopolymer
microsatellite
regions.
[128] FIG. 29 shows that MLHldn offers a method to increase prime editing
efficiency at sites
without good ngRNAs, such as HEK4
[129] FIG. 30 shows that MLHldn improves PE at disease sites.
[130] FIG. 31 shows that MLHIdn enhances installation of the protective APOE
Christchurch
allele in mouse astrocytes. A 50% boost in editing efficiency and a large
reduction in indels is
shown.
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[131] FIG. 32 shows that HEK293T cells are MMR-compromised. The MLH1 promoter
is
hypermethylated in HEF293T, resulting in lower MUD. expression.
[132] FIGs. 33A-33B show that MLH1 dn enhances prime editing in HeLa cells.
FIG. 33A
shows prime editing with PE2. FIG. 338 shows prime editing with PE3.
[133] FIGs. 34A-34B show that MLHldn enhances prime editing in HeLa cells.
FIG. 34A
shows editing of PRNP +6 G to T. FIG. 34B shows editing of APOE +6 G to T and
+10 C to A.
[134] FIGs. 35A-35B show that MtHldn has a larger effect in MMR competent cell
lines like
HeLa.
[135] FIGs. 36A-36D show that MLI-Ildn improvements synergize with stabilized
pegRNAs.
[136] FIGs. 37A-37B show that contiguous substitutions are useful as another
strategy for
evading MMR.
[137] FIG. 38 shows that MMR does not efficiently repair 3 or more contiguous
substitutions.
Contiguous substitutions therefore offer a method for circumventing MMR and
boosting PE
efficiency.
[138] FIGs. 39A-39C show that MLH1neg improves PE in HeLa cells.
[139] FIGs. 40A-40G show that pooled Repair-seq CRISPRi screens reveal genetic

determinants of substitution on prime editing outcomes. FIG. 40A shows that
prime editing with
the PE2 system is mediated by the PE2 enzyme (Streptococcus pyogenes Cas9
(SpCas9) H840A
nickase fused to a reverse transcriptase) and a prime editing guide RNA
(pegRNA). The PE3
system uses an additional single guide RNA (sgRNA) to nick the non-edited
strand and yield
higher editing efficiency. PBS, primer binding site. RT template, reverse
transcription template.
FIG. 40B provides an overview of prime editing Repair-seq CRISPRi screens. A
library of
CRISPRi sgRNAs and a pre-validated prime edit site are transduced into CRISPRi
cell lines and
transfected with prime editors targeting the edit site. CRISPRi sgRNA
identities and prime edited
sites are amplified together from genomic DNA and paired-end sequenced
together to link each
genetic perturbation with editing outcome. SaCas9, Staphylococcus aureus Cas9.
FIG. 40C
shows the effect of each CRISPRi sgRNA on the percentage of sequencing reads
reporting the
intended CrC-to-C=G prime edit at the targeted edit site in pooled CRISPRi
screens. Each value
depicts all sequencing reads carrying the same CR1SPRi sgRNA. FIG. 401) shows
the effect of
CRISPRi sgRNAs on editing efficiency in all screen conditions. Black dots
represent individual
non-targeting sgRNAs, black lines show the mean of all non-targeting sgRNAs,
and gray
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shading represents kernel density estimates of the distributions of all
sgRNAs. FIGs. 40E-40G
show comparisons of gene-level effects of CRISPRi targeting on the intended GC-
to-C=G prime
edit across different screen conditions. (FIG. 40E) K562 PE2 vs. HeLa PE2.
(FIG. 40F) K562
PE3+50 vs. HeLa PE3+50. (FIG. 40G) 1K562 PE2 vs. K562 PE3+50. The effect of
each gene is
calculated as the average log2 fold change in frequency from non-targeting
sgRNAs for the two
most extreme sgRNAs targeting the gene. Plotted quantities are the mean of n=2
independent
biological replicates for each cell type, with bars showing the range of
values spanned by the
replicates. Black dots represent 20 random sets of three non-targeting sgRNAs.
11401 FIGs. 41A-41J show genetic modulators of unintended prime editing
outcomes. FIGs.
41A-41D show representative examples of four categories of unintended prime
editing outcomes
observed in CRISPRi screens. In each panel, the black bar depicts the sequence
of an editing
outcome, the blue bar depicts genomic sequence around the targeted editing
site, and the orange
bar depicts the pegRNA sequence. Blue and orange lines between the editing
outcome and the
genome or pegRNA depict local alignments between the outcome sequence and the
relevant
reference sequence. Mismatches in alignments are marked by X's, and insertions
are marked by
downward dimples. The location of the programmed edit is marked by a grey box.
Red and cyan
rectangles on the genome mark SaCas9 protospacers and PAMs, and black vertical
lines mark
the locations of SaCas9 nick sites. Orange, beige, grey, and red rectangles on
the pegRNA mark
the primer binding site (PBS), reverse transcription template (RTT), scaffold,
and spacer,
respectively. FIGs. 41E-41F provide a summary of editing outcome categories
observed in PE2
screens (FIG. 41E) and in PE3+50 screens (FIG. 41F) in K562 cells. Plotted
quantities are the
mean SD of all sgRNAs for each indicated gene (60 non-targeting sgRNAs,
three sgRNAs per
targeted gene), averaged across n=2 independent biological replicates. FIGs.
41G-4111 show a
comparison of the effects of knockdown of all genes targeted in CRISPRi
screens on the
frequency of joining of reverse transcribed sequence at unintended locations
(FIG. 41G) or the
frequency of deletions (FIG. 4111) from PE3+50. The effect of each gene is
calculated as the
average 1og2 fold change in frequency from non-targeting sgRNAs for the two
most extreme
sgRNAs targeting the gene. Plotted quantities are the mean of n=2 independent
biological
replicates for each cell type, with bars showing the range of values spanned
by the replicates.
Black dots represent 20 random sets of three non-targeting sgRNAs. FIG. 411
shows the
frequency of deletion as a function of genomic position relative to programmed
PE3+50 nicks
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(dashed vertical lines) in K562 screen replicate 1 across all reads for
indicated sets of CRISPRi
sgRNAs (black line: 60 non-targeting sgRNAs; orange and green lines: three
sgRNAs targeting
each of MSH2, MSH6, MLH1, and PMS2) (top). Log2 fold change in frequency of
deletion as a
function of genomic position from MSH2, MSH6, IMLHI1, and PMS2 sgRNAs compared
to non-
targeting sgRNAs (bottom). FIG. 41 J shows the effect of gene knockdowns on
the fraction of
all observed deletions that remove sequence at least 25-nt outside of
programmed PE3+50 nicks
in K562 screens. Each dot represents all reads for all sgRNAs targeting each
gene. Black dots
represent 20 sets of three random non-targeting sgRNAs.
11411 FIGs. 42A-42D show a model for mismatch repair of prime editing
intermediates. FIG.
42A shows a model for DNA mismatch repair (MMR) of PE2 intermediates. MMR.
excises and
replaces the nicked strand during repair of the prime editor-generated
heteroduplex substrate.
Infrequent ligation of the nick before MMR recognition deprives the strand
discrimination signal
for MMR, resulting in un-biased resolution of the heteroduplex. FIG. 42B shows
a model for
MMR of PE3 intermediates. PE3 installs an additional nick on the non-edited
strand that can
direct MMR to replace the non-edited strand. Ligation of the edited strand
nick leaves only the
complementary-strand nick to signal repair by MMR, resulting in the desired
prime editing
outcome. FIG. 42C shows prime editing efficiencies of PE2 and PE3 prime
editors at
endogenous sites (HEK3, EMX1, and MINX]) in HEK293T cells pre-treated with
knockdown
siRNAs against MSH2, MSH6, MLH1, or PMS2 transcripts. Cells were pre-
transfected with
siRNAs 3 days prior to transfection with prime editor components and siRNAs.
Genomic DNA
was harvested 3 days following transfection with prime editors and additional
siRNA, then
sequenced. Bars represent the mean of n=3 independent biological replicates.
FIG. 42D shows
prime editing efficiencies in HAP! AMSH2 and HAP! AMLH1 cells (mean of n=3
independent
biological replicates). A, gene knockout.
11421 F1Gs. 43A-43F show that engineered dominant negative MMR proteins
(dominant
negative variants of MSH2, MSH6, PMS2, and MLII1) enhance prime editing. FIG.
43A shows
editing improvement at HEK2, ENLY1, and MINX] sites by co-expression of PE2 in
trans with
human MMR proteins or dominant negative variants in HEK293T cells. MMR
proteins include
MSH2, MSH6, PM S2, and MLH1. Dominant negative variants are designated as MSH2
K675R,
MSH6 K1 140R, PMS2 E41A, PMS2 E705K, MLH1 E34A, and MLH1 A756. All values from
n
=3 independent biological replicates are shown. FIG. 43B shows functional
annotation of the
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756-aa human MLH1 protein, including an ATPase domain, MSH2 interaction
domain, NLS
domain, PMS2 dimerization domain, and an endonuclease domain. FIG. 43C shows
editing
enhancement of MLIT1 variants co-expressed with PE2 in HEK293T cells at HIM,
ENIX1, and
Rti7VX/ sites. Red boxes indicate mutations that inactivate MLH1 ATPase or
endonuclease
function. MLHldn, MLH1 A754-756. MLH1NTD¨NLS, codon-optimized MLH1 1-335¨
NLSSV40. All values from n=3 independent biological replicates are shown. FIG.
43D shows a
comparison of the top three dominant negative MLH1 variants at additional
prime edits. All
values from n=3 independent biological replicates are shown. FIG. 43E shows
prime editing
with PE2 and MLHldn in trans, PE2 and MLH1NTD¨NLS in trans, and PE2¨P2A¨MLH1dn

(human codon optimized) in HEK293T cells. Bars represent the mean of n = 3
independent
biological replicates. FIG. 43F compares the structure of PE2, PE3, PE4, and
PE5. In particular,
the PE4 editing system consists of a prime editor enzyme (nickase Cas9-RT
fusion), MLHldn,
and pegRNA. The PE5 editing system consists of a prime editor enzyme, MLHldn,
pegRNA,
and second-strand nicking sgRNA. FIG. 43G shows editing efficiencies of PE2,
PE3, PE4, and
PE5 systems in HEK293T cells. Bars represent the mean of n = 3 independent
biological
replicates).
[143] FIGs. 44A-44G show the characterization of PE4 and PE5 across diverse
prime editing
classes and cell types. FIG. 44A provides a summary of prime editing
enhancement by PE4 and
PE5 compared to PE2 and PE3 for 84 single-base substitution edits (seven for
each substitution
type) across seven endogenous sites in HEK293T cells. The grand mean SD of
all individual
values of n = 3 independent biological replicates are shown. FIG. 44B shows
installation of
single base mutations at the FANCF locus with PE2, PE3, PE4, and PE5 in
HEK293T cells. Bars
represent the mean of n =3 independent biological replicates. FIG. 44C shows
that PE4
improves the 1- and 3-bp insertion and deletion prime edits compared to PE2 in
HEK293T cells.
Bars represent the mean of n = 3 independent biological replicates. FIG. 44D
shows PE4 editing
enhancement over PE2 across 33 different insertion and deletion prime edits.
Bars represent the
mean of all individual values of n=3 independent biological replicates. FIGs.
44E-44F provide a
summary of PE2 and PE4 editing efficiencies for 35 different substitutions of
1 to 5 contiguous
bases at five endogenous sites in HEK293T cells. Seven pegRNAs were tested for
each number
of contiguous bases altered. The mean SD of all individual values of n = 3
independent
biological replicates are shown. FIGs. 44G-441I show that installation of
additional silent or
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benign mutations near the intended edit can increase editing efficiency by
generating a
heteroduplex substrate that evades MMR. The PAM sequence (NGG) for each target
is
underlined. The amino acid sequence of the targeted gene is centered above
each DNA codon.
Values represent the mean SD of n=3 independent biological replicates. FIG.
441 shows a
comparison of prime editing enhancement in different cell types. PE4 and PE5
systems enhance
prime editing to a greater extent in MMR deficient cells (MMR-) than in MMR
proficient cells
(MMR+). The same set of 30 pegRNAs encoding single-base substitution edits
were tested in
HEK293T and HeLa cells. K562 and U2OS cells were edited with 10 pegRNAs that
are a direct
subset of the 30 pegRNAs tested in HEK293T and HeLa cells. The mean SD of
all individual
values of sets of n = 3 independent biological replicates are shown. P values
were calculated
using the Mann-Whitney U test. FIG. 44J shows prime editing with PE2, PE3,
PE4, and PE5 in
HeLa, K562, and U2OS cells. Bars represent the mean of n =3 independent
biological
replicates).
[144] FIGs. 45A-4511 show the effect of dominant negative MLH1 on prime
editing product
purity and off-targeting. FIG. 45A shows that edit-encoding pegRNAs program a
base change
within the nascent 3' DNA flap and generate a heteroduplex following flap
interconversion. Non-
editing pegRNAs template a 3' DNA flap with perfect complementarity to the
genomic target
site. FIG. 45B shows the frequency of indels from PE3 or PE5 with four edit-
encoding
pegRNAs that program single base mutations or four non-editing pegRNAs. Short
horizontal
bars indicate the mean of all individual values of sets of n = 3 independent
biological replicates.
FIG. 45C shows the ratio of indel frequency from PE5 over PE3 with 4 edit-
encoding pegRNAs
that program single base mutations or four edit-encoding pegRNAs that program
single base
mutations or four non-editing pegRNAs. Short horizontal bars indicate the mean
of all individual
values of sets of n =3 independent biological replicates. FIG. 45D shows
distribution of
deletions at genomic target DNA formed by PE3 and PE5 using 12 substitution-
encoding
pegRNAs at endogenous DNMT1 and RNF2 loci in HEK293T cells. Dotted lines
indicate
position of pegRNA- and sgRNA-directed nicks. Data represent the mean SD of
n=3
independent biological replicates. FIG. 45E shows PE5/PE3 ratio of frequency
of deletions that
remove sequence greater than 25-nt outside of pegRNA- and sgRNA-directed nicks
in HEK293T
cells. Each dot represents one of 84 total pegRNAs that program substitution
edits at a combined
seven loci (mean of n=3 independent biological replicates). FIG. 45F shows
PE5/PE3 ratio of
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frequency of editing outcomes with unintended pegRNA scaffold sequence
incorporation or
unintended flap rejoining in HEK293T cells. Each dot represents one of 84
total pegRNAs that
program substitution edits at a combined seven loci (mean of n = 3 independent
biological
replicates). FIG. 45G shows off-target prime editing by IPE2 and PE4 in
HEK293T cells. Bars
represent the mean of n =3 independent biological replicates). FIG. 45H shows
high-throughput
sequencing analysis of 17 sensitive microsatellite repeat loci used for
clinical diagnosis of MMR
deficiency. HAP1 and HeLa cells are MMR-proficient, and HCT116 cells have
impaired MMR.
HAP1 AMSH2 cells underwent 60 cell divisions following MSH2 knockout. HeLa
cells were
transiently transfected with PE2 or PE4 components and incubated for 3 days
before sequencing.
wt, wild-type. All values from n=2 independent biological replicates are
shown.
[145] FIGs. 46A-46F show that PEmax architecture with PE4 and PE5 editing
systems
enhances editing at disease-relevant gene targets and cell types. FIG 46A
shows a schematic of
PE2 and PEmax editor architectures. bpNLSSV40, bipartite SV40 NLS nuclear
localization
signal. MMLV RT, Moloney Murine Leukemia Virus reverse transcriptase
pentamutant; codon
opt., human codon-optimized. FIG. 46B shows that engineered pegRNAs (epegRNAs)
contain a
3' RNA structural motif that improve prime editing performance. FIG. 46C shows
prime editing
efficiencies of PE4 and PE5 combined with PEmax architectures and epegRNAs.
Seven single-
base substitution edits targeting different loci were tested in HeLa and
HEK293T cells. Fold
changes indicate the average of fold increases from each edit tested. The
meand-SD of all
individual values of n=3 independent biological replicates are shown. FIG. 46D
shows prime
editing at therapeutically-relevant sites in wild-type HeLa and HEK293T cells.
The HBB locus is
edited at the E6 codon commonly mutated in patients with sickle cell disease
(E6V). The
CDKL5 edit is at a site for which the c.1412delA mutation causes CDKL5
deficiency disorder.
epegRNAs were used for editing the HBB, PRNP, and CDKL5 loci. Bars represent
the mean of
n=3 independent biological replicates. FIG. 46E shows correction of CDKL5
c.1412delA via an
A=T insertion and a silent G=C-to-AT edit in iPSCs derived from a patient
heterozygous for the
allele. Editing efficiencies indicate the percentage of sequencing reads with
c.1412delA
correction out of editable alleles that carry the mutation. lndel frequencies
reflect all sequencing
reads that contain any indels. Bars represent the mean of n=3 independent
biological replicates.
FIG. 46F shows prime editing in primary human T cells. Bars represent the mean
of n=3
independent biological replicates from different healthy T cell donors.
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[146] FIGs. 47A-47J show the design and results of Repair-seq screens for
substitution prime
editing outcomes. FIG. 47A shows optimization of a Staphylococcus aureus (Sa)-
pegRNA for
installation of a G=C-to-C=G edit within a lentivirally integrated HBB
sequence using SaPE2 in
HEK293T cells. PBS, primer-binding site. Data represent the mean of n =3
independent
biological replicates. FIG. 47B shows the design of the prime editing Repair-
seq lentiviral vector
(pPC1000). In Repair-seq screens, a 453-bp region containing CRISPRi sgRNA
sequence and
prime editing outcome is amplified from genomic DNA for paired-end Illumina
sequencing. The
CRISPRi sgRNA is sequenced with a 44-nt Illumina forward read (R1), and the
prime edited site
(including +50 and ¨50 nick sites) is sequenced with a 263-nt Illumina reverse
read (R2). Black
triangles indicate positions of SaPE2-induced nicks programmed by Sa-pegRNA
and Sa-
sgRNAs. Sizes of all vector components are to scale. FIG. 47C shows a
schematic of PE2,
PE3+50, and PE3-50 prime editing configurations with SaPE2 protein (SaCas9
N580A fused to
an engineered MMLV RT). FIG. 47D shows validation of intended CrC-to-C,G
editing at the
lentivirally-integrated Repair-seq edit site in HeLa cells expressing
dCas9¨BFP¨KRAB cells.
Bars represent the mean of n =2 independent biological replicates. FIG. 47E
shows prime
editing at the Repair-seq edit site with and without blasticidin selection in
HeLa cells expressing
dCas9¨BFP¨KRAB. SaPE2¨P2A¨BlastR prime editor was used for all conditions.
Bars
represent the mean of n =2. FIG. 47F shows functional annotation classes of
the genes targeted
by the pooled CRISPRi sgRNA library used in Repair-seq screens. FIGs. 47G-47J
show that the
knockdown of MSH2, MSH6, MLH1, and PMS2 increases the frequency of the
intended +6
GC-to-C=G prime edit in all Repair-seq screens. Dots represent reads from
individual CRISPRi
sgRNAs.
[147] FIGs. 48A-48I show the genetic modulators of unintended prime editing
outcomes. FIG.
48A shows an overview of PE3-50 outcomes in HeLa CRISPRi screens. TP53BP1
knockdown
dramatically reduces formation of all unintended editing outcomes. FIG. 48B
shows additional
details of PE2 outcomes in K562 CRISPRi screens, supplementing FIG. 41H. FIG.
48C shows
additional details of PE3+50 outcomes in K562 CRISPRi screens, supplementing
information in
FIG. 41G. FIGs. 48D-48I show comparisons of effects of gene knockdown on
frequencies of
indicated outcome categories in indicated screen conditions. Platted
quantities are the mean of
the 1og2 fold changes from non-targeting sgRNAs for the two most extreme
sgRNAs per gene,
averaged over n =2 independent biological replicates per condition. Error bars
mark the range of
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values spanned by the replicates. Black dots represent 20 random sets of three
non-targeting
sgRNAs. FIG. 48D shows that MSH2, MLH1, and PMS2 knockdown produce larger fold

changes in installation of additional edits than in intended edits in K562 PE2
screens. FIG. 48E
shows unintended joining of reverse transcribed sequence in PE2 screens in
K562 and HeLa cells
are most increased by knockdown of Fanconi anemia genes (red) as well as a set
of RAD51
homologs and other genes involved in homologous recombination (blue). FIG.
48:F shows
deletions in in PE2 screens in K562 and HeLa cells are most increased by a set
of RAD51
homologs and other genes involved in homologous recombination (blue). FIG. 48G
shows that
in addition to MSH2, MLH1, and PMS2, HLTF knockdown produces larger fold
changes in
installation of additional edits than in intended edits in K562 PE3+50
screens. FIG. 48111 shows
that tandem duplications in HeLa and K562 PE3+50 screens are most decreased by
knockdown
of FOLD and RFC subunits. FIG. 481 shows deletions in HeLa PE3+50 and PE3-50
screens
have dramatically divergent genetic regulators, highlighting differences in
the processing of the
different overhang configurations.
1148] FIGs. 49A-49F show validation of prime editing Repair-seq screen
results. FIGs. 49A-
49B show alignment of Sa-pegRNAs, their templated 3' DNA flaps following SaPE2
reverse
transcription, and the genomic target sequence (top). Compared to the Sa-
pegRNA used in
Repair-seq screens (FIG. 49A), an Sa-pegRNA with recoded scaffold sequence
(FIG. 49B)
templates an extended 3' DNA flap with reduced homology with genomic target
sequence. The
recoded Sa-pegRNA contains 2 base pair changes that preserve base pairing
interactions within
the scaffold. Reverse transcription of the Sa-pegRNA scaffold can generate a
misextended 3' flap
that is incorporated into the genome. Vertical lines depict base pairing. X's
depict mismatches
between the misextended reverse-transcribed 3' flap and genomic sequence.
FIGs. 49A-49B also
show frequencies of editing outcome categories observed at the screen edit
site from arrayed PE
and PE3+50 experiments in HeLa CRISPRi cells (bottom). Prime editing with the
Sa-pegRNA
used in siteRepair-seq screens (FIG. 49A) or a recoded Sa-pegRNA (FIG. 49B)
results in
different frequencies of installation of unintended edits from nearly-matched
scaffold. Plotted
quantities are the meand.,SD of n=4 independent biological replicates, for
each cell line
containing MSH2 or non-targeting CR1SPRi sgRNAs. FIG. 49C shows the mechanism
of DNA
mismatch repair in humans. FIG. 49D shows mismatch repair of a prime editing
heteroduplex
intermediate could install additional non-programmed nicks from MutLa
endonuclease activity.
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Excision from these non-programmed nicks and subsequent repair of the
resulting intermediates
may contribute to larger and more frequent indel byproducts observed from MMR
activity. FIG.
49E shows the knockdown efficiency of siRNA treatment relative to a non-
targeting siRNA
control in HEK293T cells. Cells were transfected with siRNAs, incubated for 3
days, transfected
with PE2, pegRNAs, and the same siRNAs, then incubated for another 3 days
before relative
RNA abundances were assayed by RT-qPCR. NT, non-targeting. Data represent the
mean of n=3
independent biological replicates. Each dot represents the mean of n = 3
technical replicates.
FIG. 49F shows editing in HEK293T cells co-transfected with prime editor
components and
siRNAs. Cells were not pre-treated with siRNAs before transfection with prime
editor. Bars
represent the mean of n =3 independent biological replicates.
[149] FIGs. 50A-50H show the development and characterization of dominant
negative MMR
proteins that enhance prime editing. FIG. 50A shows the prime editing
efficiencies from MMR
proteins or dominant negative variants expressed in trans with or fused
directly to PE2 in
HEK293T cells. 32aa linker, (SGGS)x2¨XTEN¨(SGGS)x2 (SEQ ID NO: 125)
(SGGSSGGSSGSETPGTSESATPES SGGSSGGS (SEQ ID NO: 125) or structurally, [SGGS}-
[SGGS]-[SGSETPGTSESATPESMSGGSMSGGS] (SEQ ID NO: 125)). codon opt., human
codon optimized. Data within the same graph originate from experiments
performed at the same
time. Data represent the mean SD of n =3 independent biological replicates.
FIG. 50B shows
titration of MLI-11 dn plasmid and PE2 plasmid transfection doses in HEK293T
cells. Maximum
plasmid amounts tested were 200 ng PE2 and 100 ng MLHIdn. Data represent the
mean SD of
n =3 independent biological replicates. FIG. 50C shows prime editing with
MLHldn co-
expression in MMR-deficient HCT116 cells that contain a biallelic deletion in
MLH1. Bars
represent the mean of 3 replicates. FIG. 50D shows a comparison of prime
editing with human
MLHidn (human codon-optimized) or mouse MLH1 dn (mouse codon optimized) in
human
HEK293T cells. Bars represent the mean of n = 3 independent biological
replicates. FIG. 50E
shows a comparison of prime editing with human MLITIdn (human codon optimized)
or mouse
MLHldn (mouse codon optimized) in mouse N2A cells. Bars represent the mean of
n = 3
replicates. FIG. 50F shows that MLIII knockout in clonal HeLa cell lines
enhances prime
editing efficiency to a greater extent than ML111 co-expression in clonal wild-
type HeLa cells. d,
knockout. Bars represent the mean of n = 3 or 4 independent biological
replicates. FIG. 50G
shows editing at the IFANCF locus with PE3b and IPE5b (complementary-strand
nick that is
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specific for the edited sequence) in HEK293T cells. PE5b, PE3b editing system
with MLHldn
co-expression. Bars represent the mean of n = 3 independent biological
replicates. FIG. 50H
shows editing at the I1EK2 locus with complementary-strand nicks in HEK293T
cells. "None"
indicates the lack of a nick, which denotes a PE2 or PE4 editing strategy.
Bars represent the
mean of n = 3 independent biological replicates.
[150] FIGs. 51A-51J show the characterization of PE4 and PE5 across diverse
prime edit
classes and cell types. FIG. 51A shows a comparison of PE2, PE3, PE4, and PE5
for 84 single-
base substitution prime edits across seven endogenous sites in HEK293T cells.
Bars represent the
mean of n =3 independent biological replicates). FIG. 51B provides a summary
of PE4
enhancement in editing efficiency over PE2 for 84 single-base substitution
edits across seven
endogenous sites in HEK293T cells. PE4/PE2 fold improvements may be lower for
PAM edits
due to the high basal editing efficiency for PAM edits or the high
representation of G=C-to-C=G
edits (five out of 15 in this category). Data represent the mean SID of n =
3 independent
biological replicates. FIG. 51C shows the efficiencies of single-base
substitution prime edits that
alter the PAM (+5 G and +6 G bases) of prime editing target protospacers in
HEK293T cells.
Four G=C-to-A=T, five G=C-to-C=G, and six G=C-to-T=A PAM edits across a
combined seven
endogenous sites are shown. The mean of all individual values of n =3
independent biological
replicates are shown. FIG. 51D shows the effect of siRNA knockdown of MMR
genes on G=C-
to-C=G editing at the RNF2 locus in HEK293T cells. Bars represent the mean of
n =3
independent biological replicates. FIG. 51E shows the effect of MMR gene
knockout on G=C-to-
C=G editing at the RNF2 locus in HAP! cells. A, gene knockout. Bars represent
the mean of n =
3 independent biological replicates. FIG. 51F shows prime editing at the
integrated screen edit
site with CRISPRi knockdown in HeLa CRISPRi cells. PE2 indicates editing with
SaPE2 protein
and Sa-pegRNA. PE3+50 indicates editing with SaPE2 protein, Sa-pegRNA, and Sa-
sgRNA that
programs a +50 complementary-strand nick. Bars represent the mean of n = 5
independent
biological replicates. FIG. 51G provides a summary of PE5 enhancement in
editing efficiency
over PE3 for 84 single-base substitution edits in HEK293T cells. The grand
mean SD of all
individual values of n = 3 independent biological replicates are shown. FIG.
5111 shows PE4
enhancement in editing efficiency over PE2 across a range of insertion and
deletion prime edit
lengths in HEK293T cells. A total of 33 different prime edits at a combined
three endogenous
loci are shown. The mean of all individual values of n =3 independent
biological replicates are
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shown. FIG. 511 shows that PE5 improves editing efficiency and reduces indel
byproducts
compared to PE3 across small insertion and deletion prime edits in HEK293T
cells. FIG. 51J
shows PE2 and PE4 editing efficiencies at 33 different insertion and deletion
prime edits across a
combined three endogenous loci. Bars represent the mean of all individual
values of n=3
independent biological replicates.
[151] FEGs. 52A-52C show characterization of PE4 and PE5 systems and improved
prime
editing efficiency with additional silent mutations. FIG. 52A shows
substitutions of contiguous
bases with PE2 and PE4 in HEK293T cells. The top sequence indicates the
original, unedited
genomic sequence. Numbers denote the position of the edited nucleotide
relative to the PE2 nick
site. Nucleotides within the SpCas9 PAM sequence (NGG) are underlined.
Sequences of the
intended edited product are shown below, with edited nucleotides marked in
red. Bars represent
the mean of n =3 independent biological replicates. FIG. 52B shows that
installation of
additional silent mutations can increase prime editing efficiency by evading
MMR. PE4/PE2
fold-change in editing frequency reflects the extent to which MMR activity
impedes the
indicated prime edit. Edited nucleotides that make the indicated coding
mutation are marked in
red, and edited nucleotides that make silent mutations are marked in green.
Data represent the
mean:ESD of n=3 independent biological replicates. FIG. 52C shows installation
of 22 single-
base substitution prime edits across seven endogenous sites in HeLa cells with
PE2, PE3, PE4,
and PE5. Bars represent the mean of n=3 independent biological replicates.
[152] FIGs. 53A-53G show the effect of dominant negative MLH1 on prime editing
product
purity and off-targeting. FIG. 53A shows the frequency of indels in HEK293T
cells treated with
pegRNAs, nicking sgRNAs, and PE2 enzyme, R'F-impaired PE2 (PE2--dRT), or
nickase Cas9
(SpCas9 H840A), with and without MLHldn. Non-editing pegRNAs encode a 3' DNA
flap with
perfect homology to the genomic target. Bars represent the mean of n =3
independent biological
replicates. FIG. 53B shows the distribution of deletion outcomes from PE3 and
PE5 at
endogenous loci in HEK293T cells. 12 different pegRNAs that program single-
base substitutions
were tested at each indicated locus. Dotted lines indicate position of pegRNA-
and sgRNA-
directed nicks. Data represent the mean a: SD of n = 3 independent biological
replicates. FIG.
53C shows the distribution of deletion outcomes from PE3 and PE5 with an edit-
encoding and
non-editing pegRNA in HEK293T cells. The non-editing pegRNA templates a 3' DNA
flap with
perfect complementarity to the genomic target sequence. Data represent the
mean SD of n =3
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independent biological replicates. FIG. 53D shows the frequency of all prime
editing outcomes
with unintended pegRNA scaffold sequence incorporation or unintended flap
rejoining in
1-1EK293T cells. 12 pegRNAs each programming a different single-base
substitution were tested
at each of the seven indicated loci. Each dot represents an individual pegRNA
at the indicated
loci (mean of n = 3 independent biological replicates). FIG. 53E shows the off-
target prime
editing by PE2 and PE4 in :HEK293T cells (mean of n =3 independent biological
replicates).
FIG. 53F shows the distribution and cumulative distribution of microsatellite
repeat lengths in
the indicated cell types and treatments. HAP1 and HeLa cells are MN/TR-
proficient, and HCT116
cells have impaired MMR. HAP! AMSH2 cells underwent 60 cell divisions
following knockout
of MSH2. HeLa cells were transiently transfected with PE2 or PE4 components
and grown for a
following 3 days before sequencing. wt, wild-type. All values from n =2
independent biological
replicates are shown. FIG. 53G shows prime editing at the on-target locus in
HeLa cells
transfected with PE2 or PE4 components. Bars represent the mean of n =2
independent
biological replicates. Microsatellite lengths were assayed from genomic DNA
taken from these
PE2 and PE4-treated HeLa cells.
[153] FIGs. 54A-54F show that use of PEmax architecture with PE4 and PE5
editing systems
enhances editing at disease-relevant gene targets and cell types. FIG. 54A
shows a schematic of
PE2 and PEmax editor architectures. bpNLSsw , bipartite SV40 NLS. MMLV RT,
Moloney
Murine Leukemia Virus reverse transuiptase pentamutant. GS codon, Genscript
human codon
optimized. FIG. 548 shows engineered pegRNAs (epegRNAs) containing a 3' RNA
structural
motif that improve prime editing performance. FIG. 54C shows prime editing
efficiencies of
PE4 and PE5 combined with PEmax architectures and epegRNAs. Seven single-base
substitution
edits targeting different loci were tested in HeLa and HEK293T cells. Fold
changes indicate the
average of fold increases from each edit tested. The mean SD of all
individual values of n =3
independent biological replicates are shown. FIG. 54D shows prime editing at
therapeutically-
relevant sites in wild-type HeLa and HEK293T cells. The HBB locus is edited at
the E6 codon
commonly mutated in patients with sickle cell disease (E6V). The CDKL5 edit is
at a site for
which the c.1412delA mutation causes CDKL5 deficiency disorder. epegRNAs were
used for
editing the HBB, PRNP, and CDKL5 loci. Bars represent the mean of n =3
independent
biological replicates. FIG. 54E shows the correction of CDKL5 c.1412delA via
an A=T insertion
and a silent GC-to-A=T edit in iPSCs derived from a patient heterozygous for
the allele. Editing
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efficiencies indicate the percentage of sequencing reads with c.1412delA
correction out of
editable alleles that carry the mutation. Indel frequencies reflect all
sequencing reads that contain
any indels. Bars represent the mean of n = 3 independent biological
replicates. FIG. 54F shows
prime editing in primary T cells. Bars represent the mean of n = 3 independent
biological
replicates from different healthy T cell donors.
[154] FIGs. 55A-55B show the development of PEmax and application of PE4 and
PE5 to
primary cell types. FIGs. 55A-55B show screening of prime editor variants to
maximize editing
efficiency in HeLa cells. All prime editor architectures carry a Cas9 H840A
mutation to prevent
nicking of the complementary DNA strand at the target protospacer. *NLSSV40
contains a 1-aa
deletion outside the PKKKRKV (SEQ ID NO: 132) NLSSV40 consensus sequence. All
individual values of n =3 independent biological replicates are shown.
[1551 FIGs. 56A-56G show development of PEmax and application of PE4 and PE5
to primary
cell types. MG. 56A shows a screen of prime editor variants for improved
editing efficiency
with the PE3 system in HeLa cells. All prime editor architectures carry a
SpCas9 H840A
mutation to prevent nicking of the complementary DNA strand at the target
protospacer.
NLSSV40 indicates the bipartite SV40 NLS. *NLSSV40 contains a 1-aa deletion
outside the
PKKKRKV (SEQ ID NO: 132) NLSSV40 consensus sequence. All individual values of
n=3
independent biological replicates are shown. FIG. 56B shows the architecture
of the original
PE2 editor (Anzalone et al., 2019), PE2* (Liu et al., 2021), CMP-PE-V1 (Park
et al., 2021), and
prime editor variants developed in this work (PEmax, CMP-PEmax). HN1, HIVIGN1;
H1G,
histone H1 central globular domain; codon opt., human codon optimized. FIG.
56C shows that
PEmax outperforms other prime editor architectures with the PE3 system in HeLa
cells. Bars
represent the mean of n=3 independent biological replicates. FIG. 56D shows
fold-change in
editing efficiency of prime editor architectures compared to PE2 with the PE3
system in HeLa
cells. The mean SD of all individual values of n=3 independent biological
replicates are shown.
FIG. 56E shows the intended editing and indel frequencies from PE4, PE4max
(PE4 editing
system with PEmax architecture), PE5, and PE5max (PE5 editing system with
PEmax
architecture) in HeLa and HEK293T cells. Seven substitution prime edits
targeting different
endogenous loci were tested for each condition. The mean SD of all
individual values of n =3
independent biological replicates are shown. FIG. 56F shows the correction of
CDKL5
c.1412delA via an .A=T insertion and a GC-to-A=T edit in iPSCs derived from a
patient
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heterozygous for the disease allele. Editing efficiencies indicate the
percentage of sequencing
reads with c.1412delA correction out of editable alleles that early the
mutation. Indel frequencies
reflect all sequencing reads that contain any indels that do not map to the
c.1412delA allele or
wild-type sequence. 1 pg of IPE2 mItNA was used in all conditions shown. Bars
represent the
mean of n = 3 independent biological replicates. FIG. 56G shows prime editing
in primary T
cells. Bars represent the mean of n = 3 independent replicates from different
T cell donors.
11561 FIGs. 57A-57B show that the recoded pegRNA scaffold reduces unintended
outcomes
from scaffold sequence incorporation. FIG. 57A shows an alignment of the prime
editing
Repair-seq target site and SaPE2-generated 3' DNA flaps templated by (top) the
Sa-pegRNA
used in Repair-seq screens, or (bottom) an Sa-pegRNA with a recoded scaffold
sequence. 3' flap
sequences are aligned with the templated region of the Sa-pegRNA shown above
(RT template
or scaffold). Red indicates position of the intended +6 CrC to C=G edit
programmed by both Sa-
pegRNAs. Blue indicates positions at which the genomic target sequence does
not align with the
3' flap sequence templated by the Sa-pegRNA scaffold. Unintended edits from
incorporation of a
3' flap containing a reverse transcribed Sa-pegRNA scaffold sequence may occur
at these blue-
indicated nucleotides. FIG. 57B shows a summary of editing outcome categories
observed in
PE2 and PE3+50 experiments in HeLa CRISPRi cells. Screen pegRNA indicates the
Sa-pegRNA
used in prime editing Repair-seq screens. Sa-pegRNA with recoded scaffold
(sequence shown in
FIG. 54A) avoids sequence homology with the Repair-seq edit site. Plotted
quantities are the
mean SD of one CRISPRi sgRNA for each indicated target (MSH2 and non-
targeting),
averaged across n =4 independent biological replicates.
[157] FIG. 58 shows a comparison of PE3max (PE3 editing system with PEmax
protein) and
PE3 (PE3 editing system with PE2 protein) in HeLa cells (mean of n = 3
independent biological
replicates).
1158] FIG. 59 shows that PE improvement with MLHldn depends on prime edit
size. MMR
most efficiently repairs substitutions and insertion and deletion errors of
fewer than or equal to
approximately 13 bp in length.
11.591 FIG. 60 shows that PEA and epegRNAs enable prime editing with a single
pegRNA
integrant.
[1601 FIG. 61 shows that PE5 improves installation of the protective
Christchurch allele in an
APOE4 mouse astrocyte model.
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[161] FIGs. 62A-62C show that inhibiting p53 enhances the efficiency and
precision of PE3
prime editing. This is particularly true when the nicking sgRNA makes a nick
upstream (- side)
of the pegRNA-directed nick. Each point on the graphs represents an individual
CRISPRi gene
knockdown in the Repair-seq screens. The axes depict 1og2 fold changes
compared to control.
Knocking down TP53BP1 (p53 gene) increases intended editing (x-axis) and
decreases three
types of unintended editing (y-axes), including joining of the reverse
transcribed sequence at
unintended locations (FIG. 62A), unintended deletions (FIG. 62B), and
unintended tandem
duplications (FIG. 62C).
[162] FIG. 63 shows that a p53 inhibitor (i53) can enhance the efficiency and
precision of PE3
prime editing. This is particularly true when the nicking sgRNA makes a nick
upstream (- side)
of the pegRNA-directed nick. Only the EMX1 site uses a nick on the "-"
side.FIG. 64 represents
various aspects of the disclosure, including the use of CRISPRi screens to
reveal cellular
genes---including mismatch repair genes¨having an impact on prime editing
outcomes, the use
of engineered MLH1 of the mismatch repair (MMR) pathway to enhance the
efficiency and
precision of prime editing, and the demonstration that improved prime editing
systems described
herein (e.g., PE4 and PE5 systems, and PEmax editor) were shown to exhibit the
same beneficial
effects in many cell types.
[163] FIG. 64 shows that CRISPRi screens reveal cellular determinants of prime
genome
editing, that engineered MLHI protein enhances prime editing efficiency and
precision, and that
improved prime editing systems were characterized across edit and cell types.
1164.1 FIG. 65 provides a schematic showing the optimization of PE2 protein.
[165] FIG. 66 shows the fold change in the frequency of the intended edit
using PE2 and
various other PE constructs in HEK293T cells (low plasmid dose) at a range of
gene targets
(HEK3, EMX1, RNF2, FANCF, FUNX , DNA/17'1, YEGFA, HEK4, PRNP, APOE, CXCR4,
HEK3).
[166] FIG. 67 shows the fold change in the frequency of the intended edit
using PE3 and
various prime editor constructs in HeLa cells at a range of gene targets
(HEK3, FANCF, RUNX1,
VEGFA).
[167] FIG. 68 shows a comparison of prime editing in I-1E1(293T vs. lieLa
editing using
various PE constructs.
[168] FIG. 69 shows NLS architecture optimization of PE3 in HeLa cells.
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[169] FIG. 70 provides a schematic showing the final PEmax construct, which
corresponds to
SEQ ID NO: 99.
[170] FIG. 71 shows that PEmax increases indels in addition to the intended
edit.
DEFINITIONS
[171] Unless defined otherwise, all technical and scientific terms used herein
have the meaning
commonly understood by a person skilled in the art to which this invention
belongs. The
following references provide one of skill with a general definition of many of
the terms used in
this invention: Singleton etal., Dictionary of Microbiology and Molecular
Biology (2nd ed.
1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988);
The Glossary
of Genetics, 5th Ed., R. Rieger etal. (eds.), Springer Verlag (1991); and Hale
& Marham, The
Harper Collins Dictionary of Biology (1991). As used herein, the following
terms have the
meanings ascribed to them unless specified otherwise.
Cas9
[172] The term "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease
comprising a
Cas9 domain, or a fragment thereof (e.g., a protein comprising an active or
inactive DNA
cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A "Cas9
domain" as used
herein, is a protein fragment comprising an active or inactive cleavage domain
of Cas9 and/or the
gRNA binding domain of Cas9. A "Cas9 protein" is a full length Cas9 protein. A
Cas9 nuclease
is also referred to sometimes as a casnl nuclease or a CRISPR (Clustered
Regularly Interspaced
Short Palindromic Repeat)-associated nuclease. CRISPR is an adaptive immune
system that
provides protection against mobile genetic elements (viruses, transposable
elements, and
conjugative plasmids). CRISPR clusters contain spacers, sequences
complementary to
antecedent mobile elements, and target invading nucleic acids. CRISPR clusters
are transcribed
and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct
processing of
pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous rib
onuclease 3 (rric),
and a Cas9 domain. The tracrRNA serves as a guide for ribonuclease 3-aided
processing of pre-
crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves a linear
or circular
dsDNA target complementary to the spacer. The target strand not complementary
to crRNA is
first cut endonucleolytically, then trimmed 3'-5' exonucleolytically. In
nature, DNA-binding and
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cleavage typically requires protein and both RNAs. However, single guide RNAs
("sgRNA", or
simply "gNRA") can be engineered to incorporate aspects of both the crRNA and
tracrRNA into
a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M.,
Doudna J.A.,
Charpentier E. Science 337:816-821(2012), the entire contents of which are
hereby incorporated
by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences
(the PAM or
protospacer adjacent motif) to help distinguish self versus non-self. Cas9
nuclease sequences and
structures are well known to those of skill in the art (see, e.g., "Complete
genome sequence of an
MI strain of Streptococcus pyogenes." Ferretti etal., J.J., McShan W.M., Ajdic
D.J., Savic D.J.,
Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S.,
Lin S.P., Qian
Y., Jia HG., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton
S.W., Roe B.A.,
McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); "CRISPR RNA

maturation by trans-encoded small RNA and host factor RNase III." Deltcheva
E., Chylinski K.,
Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J.,
Charpentier E.,
Nature 471:602-607(2011); and "A programmable dual-RNA-guided DNA endonuclease
in
adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M.,
IDoudna j.A.,
Charpentier E. Science 337:816-821(2012), the entire contents of each of which
are incorporated
herein by reference). Cas9 orthologs have been described in various species,
including, but not
limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9
nucleases and sequences
will be apparent to those of skill in the art based on this disclosure, and
such Cas9 nucleases and
sequences include Cas9 sequences from the organisms and loci disclosed in
Chylinski, Rhun,
and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas
immunity systems"
(2013) RNA Biology 10:5, 726-737; the entire contents of which are
incorporated herein by
reference. In some embodiments, a Cas9 nuclease comprises one or more
mutations that partially
impair or inactivate the DNA cleavage domain.
[173] A nuclease-inactivated Cas9 domain may interchangeably be referred to as
a "dCas9"
protein (for nuclease-"dead" Cas9). Methods for generating a Cas9 domain (or a
fragment
thereof) having an inactive DNA cleavage domain are known (see, e.g., Jinek et
al., Science.
337:816-821(2012); Qi etal., "Repurposing CRISPR as an RNA-Guided Platform for
Sequence-
Specific Control of Gene Expression" (2013) Cell. 28;152(5):1173-83, the
entire contents of
each of which are incorporated herein by reference). For example, the DNA
cleavage domain of
Cas9 is known to include two subdomains, the HNH nuclease subdomain and the
RuvC1
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subdomain. The IINH subdomain cleaves the strand complementary to the gRNA,
whereas the
RuvC1 subdomain cleaves the non-complementary strand. Mutations within these
subdomains
can silence the nuclease activity of Cas9. For example, the mutations Di OA
and 11840A
completely inactivate the nuclease activity of S. pyogenes Cas9 (jinek etal.,
Science. 337:816-
821(2012); Qi etal., Cell. 28;152(5):1173-83 (2013)). In some embodiments,
proteins
comprising fragments of Cas9 are provided. For example, in some embodiments, a
protein
comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2)
the DNA
cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or
fragments thereof
are referred to as "Cas9 variants." A Cas9 variant shares homology to Cas9, or
a fragment
thereof. For example, a Cas9 variant is at least about 70% identical, at least
about 80% identical,
at least about 90% identical, at least about 95% identical, at least about 96%
identical, at least
about 97% identical, at least about 98% identical, at least about 99%
identical, at least about
99.5% identical, at least about 99.8% identical, or at least about 99.9%
identical to wild type
Cas9 (e.g., SpCas9 of SEQ ID NO: 2). In some embodiments, the Cas9 variant may
have 1, 2, 3,
4, 5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24,
25, 26, 27, 28, 29,30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or
more amino acid
changes compared to wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 2). In some
embodiments, the
Cas9 variant comprises a fragment of SEQ ID NO: 2 Cas9 (e.g., a gRNA binding
domain or a
DNA-cleavage domain), such that the fragment is at least about 70% identical,
at least about
80% identical, at least about 90% identical, at least about 95% identical, at
least about 96%
identical, at least about 97% identical, at least about 98% identical, at
least about 99% identical,
at least about 99.5% identical, or at least about 99.9% identical to the
corresponding fragment of
wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 2). In some embodiments, the
fragment is at least
30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95% identical,
at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of
the amino acid length
of a corresponding wild type Cas9 (e.g., SpCas9 of SEQ ID NO: 2).
Circular perm utant
[174] As used herein, the term "circular permutant" refers to a protein or
polypeptide (e.g., a
Cas9) comprising a circular permutation, which is a change in the protein's
structural
configuration involving a change in the order of amino acids appearing in the
protein's amino
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acid sequence. In other words, circular perrnutants are proteins that have
altered N- and C-
termini as compared to a wild-type counterpart, e.g., the wild-type C-terminal
half of a protein
becomes the new N-terminal half. Circular permutation (or CP) is essentially
the topological
rearrangement of a protein's primary sequence, connecting its N- and C-
terminus, often with a
peptide linker, while concurrently splitting its sequence at a different
position to create new,
adjacent N- and C-termini. The result is a protein structure with different
connectivity, but which
often can have the same overall similar three-dimensional (3D) shape, and
possibly include
improved or altered characteristics, including reduced proteolytic
susceptibility, improved
catalytic activity, altered substrate or ligand binding, and/or improved
thermostability. Circular
permutant proteins can occur in nature (e.g., concanavalin A and lectin). In
addition, circular
permutation can occur as a result of posttranslational modifications or may be
engineered using
recombinant techniques.
Circularly permuted Cas9
11751 The term "circularly permuted Cas9" refers to any Cas9 protein, or
variant thereof, that
occurs as a circular permutant, whereby its N- and C-termini have been
topically rearranged.
Such circularly permuted Cas9 proteins ("CP-Cas9"), or variants thereof,
retain the ability to
bind DNA when complexed with a guide RNA (gRNA). See, Oakes etal., "Protein
Engineering
of Cas9 for enhanced function," Methods Enzymol, 2014, 546: 491-511 and Oakes
et al.,
"CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome
Modification,"
Cell, January 10, 2019, 176: 254-267, each of which are incorporated herein by
reference. The
instant disclosure contemplates any previously known CP-Cas9 or use of a new
CP-Cas9 so long
as the resulting circularly permuted protein retains the ability to bind DNA
when complexed with
a guide RNA (gRNA). Exemplary CP-Cas9 proteins are SEQ ID NOs: 54-63.
CRISPR
11761 CRISPR is a family of DNA sequences (i.e., CRISPR clusters) in bacteria
and archaea
that represent snippets of prior infections by a virus that have invaded the
prokaryote. The
snippets of DNA are used by the prokaryotic cell to detect and destroy DNA
from subsequent
attacks by similar viruses and effectively compose, along with an array of
CRISPR-associated
proteins (including Cas9 and homologs thereof) and CRISPR-associated RNA, a
prokaryotic
immune defense system. In nature, CRISPR clusters are transcribed and
processed into CRISPR
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RNA (crRNA). In certain types of CRISPR systems (e.g., type II CRISPR
systems), correct
processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA),
endogenous
ribonuclease 3 (me) and a Cas9 protein. The tracrRNA serves as a guide for
ribonuclease 3-aided
processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically
cleaves a
linear or circular dsDNA target complementary to the RNA. Specifically, the
target strand not
complementary to crRNA is first cut endonucleolytically, then trimmed 3'-5'
exonucleolytically.
In nature, DNA-binding and cleavage typically requires protein and both RNAs.
However, single
guide RNAs ("sgRNA", or simply "gNRA") can be engineered so as to incorporate
aspects of
both the crRNA and tracrRNA into a single RNA species ¨the guide RNA. See,
e.g., Jinek M.,
Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science
337:816-821(2012), the
entire contents of which is hereby incorporated by reference. Cas9 recognizes
a short motif in the
CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help
distinguish self
versus non-self. CRISPR biology, as well as Cas9 nuclease sequences and
structures are well
known to those of skill in the art (see, e.g., "Complete genome sequence of an
M1 strain of
Streptococcus pyogenes." Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic
D.J., Savic G.,
Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P.,
Qian Y., Jia H.G.,
Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe
B.A., McLaughlin
R.E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); "CRISPR RNA maturation
by trans-
encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K.,
Sharma C.M.,
Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E.,
Nature 471:602-
607(2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive
bacterial
immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A.,
Charpentier E. Science
337:816-821(2012), the entire contents of each of which are incorporated
herein by reference).
Cas9 orthologs have been described in various species, including, but not
limited to, S. pyagenes
and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be
apparent to those
of skill in the art based on this disclosure, and such Cas9 nucleases and
sequences include Cas9
sequences from the organisms and loci disclosed in Chylinski, Rhun, and
Charpentier, "The
tracrRNA and Cas9 families of type 11 CRISPR-Cas immunity systems" (2013) RNA
Biology
10:5, 726-737; the entire contents of which are incorporated herein by
reference.
11771 In certain types of CRISPR systems (e.g., type II CRISPR systems),
correct processing of
pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous
ribonuclease 3 (rnc),
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and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided
processing of pre-
crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves a linear
or circular
nucleic acid target complementary to the RNA. Specifically, the target strand
not complementary
to crRNA is first cut endonucleolytically, then trimmed 3`-5'
exonucleolytically. In nature, DNA-
binding and cleavage typically requires protein and both RNAs. However, single
guide RNAs
("sgRNA", or simply "gRNA") can be engineered to incorporate embodiments of
both the
crRNA and tracrRNA into a single RNA species¨the guide RNA.
11781 In general, a "CRISPR system" refers collectively to transcripts and
other elements
involved in the expression of or directing the activity of CRISPR-associated
("Cm") genes,
including sequences encoding a Cas gene, a tracr (trans-activating CRISPR)
sequence (e.g.
tracrRNA or an active partial tracrRNA), a tracr mate sequence (encompassing a
"direct repeat"
and a tracrRNA-processed partial direct repeat in the context of an endogenous
CRISPR system),
a guide sequence (also referred to as a "spacer" in the context of an
endogenous CRISPR
system), or other sequences and transcripts from a CRISPR locus. The tracrRNA
of the system is
complementary (fully or partially) to the tracr mate sequence present on the
guide RNA.
DNA synthesis template
[179] As used herein, the term "DNA synthesis template" refers to the region
or portion of the
extension arm of a PEgRNA that is utilized as a template strand by a
polymerase of a prime
editor to encode a 3' single-strand DNA flap that contains the desired edit
and which then,
through the mechanism of prime editing, replaces the corresponding endogenous
strand of DNA
at the target site. The extension arm, including the DNA synthesis template,
may be comprised of
DNA or RNA. In the case of RNA, the polymerase of the prime editor can be an
RNA-dependent
DNA polymerase (e.g., a reverse transcriptase). In the case of DNA, the
polymerase of the prime
editor can be a DNA-dependent DNA polymerase. In various embodiments the DNA
synthesis
template may comprise the "edit template" and the "homology arm", and all or a
portion of the
optional 5' end modifier region, e2. That is, depending on the nature of the
e2 region (e.g.,
whether it includes a hairpin, toeloop, or stem/loop secondary structure), the
polymerase may
encode none, some, or all of the e2 region as well. Said another way, in the
case of a 3' extension
arm, the DNA synthesis template can include the portion of the extension arm
that spans from
the 5' end of the primer binding site (PBS) to 3' end of the gRNA core that
may operate as a
template for the synthesis of a single-strand of DNA by a polymerase (e.g., a
reverse
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ftanscriptase). In the case of a 5' extension arm, the DNA synthesis template
can include the
portion of the extension arm that spans from the 5' end of the PEgRNA molecule
to the 3' end of
the edit template. In some embodiments, the DNA synthesis template excludes
the primer
binding site (PBS) of PEgRNAs either having a 3' extension arm or a 5'
extension arm. Certain
embodiments described here refer to an "an RT template," which is inclusive of
the edit template
and the homology arm, i.e., the sequence of the PEgRNA extension arm which is
actually used
as a template during DNA synthesis. The term "RT template" is equivalent to
the term "DNA
synthesis template." In certain embodiments, an RT template may be used to
refer to a template
polynucleotide for reverse transcription, e.g., in a prime editing system,
complex, or method
using a prime editor having a polymerase that is a reverse transcriptase. In
some embodiments, a
DNA synthesis template may be used to refer to a template polynucleotide for
DNA
polymerization, e.g., RNA-dependent DNA polymerization or DNA-dependent DNA
polymerization, e.g., in a prime editing system, complex, or method using a
prime editor having
a polymerase that is an RNA-dependent DNA polymerase or a DNA-dependent DNA
polymerase.
[180] In some embodiments, the DNA synthesis template is a single-stranded
portion of the
PEgRNA that is 5' of the PBS and comprises a region of complementarity to the
PAM strand
(i.e., the non-target strand or the edit strand), and comprises one or more
nucleotide edits
compared to the endogenous sequence of the double stranded target DNA. In some

embodiments, the DNA synthesis template is complementary or substantially
complementary to
a sequence on the non-target strand that is downstream of a nick site, except
for one or more
non-complementary nucleotides at the intended nucleotide edit positions. In
some embodiments,
the DNA synthesis template is complementary or substantially complementary to
a sequence on
the non-target strand that is immediately downstream (i.e., directly
downstream) of a nick site,
except for one or more non-complementary nucleotides at the intended
nucleotide edit positions.
In some embodiments, one or more of the non-complementary nucleotides at the
intended
nucleotide edit positions are immediately downstream of a nick site. In some
embodiments, the
DNA synthesis template comprises one or more nucleotide edits relative to the
double-stranded
target DNA sequence. In some embodiments, the DNA synthesis template comprises
one or
more nucleotide edits relative to the non-target strand of the double-stranded
target DNA
sequence. For each PEgRNA described herein, a nick site is characteristic of
the particular
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napDNAbp to which the gRNA core of the PEgRNA associates, and is
characteristic of the
particular PAM required for recognition and function of the napDNAbp. For
example, for a
PEgRNA that comprises a gRNA core that associates with a SpCas9, the nick site
in the
phosphocliester bond between bases three ("-3" position relative to the
position 1 of the PAM
sequence) and four ("-4" position relative to position 1 of the PAM sequence).
In some
embodiments, the DNA synthesis template and the primer binding site are
immediately adjacent
to each other. The terms "nucleotide edit", "nucleotide change", "desired
nucleotide change",
and "desired nucleotide edit" are used interchangeably to refer to a specific
nucleotide edit, e.g.,
a specific deletion of one or more nucleotides, a specific insertion of one or
more nucleotides, a
specific substitution (or multiple substitutions) of one or more nucleotides,
or a combination
thereof, at a specific position in a DNA synthesis template of a PEgRNA to be
incorporated in a
target DNA sequence. In some embodiments, the DNA synthesis template comprises
more than
one nucleotide edit relative to the double-stranded target DNA sequence. In
such embodiments,
each nucleotide edit is a specific nucleotide edit at a specific position in
the DNA synthesis
template, each nucleotide edit is at a different specific position relative to
any of the other
nucleotide edits in the DNA synthesis template, and each nucleotide edit is
independently
selected from a specific deletion of one or more nucleotides, a specific
insertion of one or more
nucleotides, a specific substitution (or multiple substitutions) of one or
more nucleotides, or a
combination thereof. A nucleotide edit may refer to the edit on the DNA
synthesis template as
compared to the sequence on the target strand of the target gene, or a
nucleotide edit may refer to
the edit encoded by the DNA synthesis template on the newly synthesized single
stranded DNA
that replaces the endogenous target DNA sequence on the non-target strand.
Dominant Negative Variant
[ 1811 The terms "dominant negative variant" and "dominant negative mutant"
refer to genes or
gene products (e.g., proteins) that comprise a mutation that results in the
gene product acting
antagonistically to the wild-type gene product (i.e., inhibiting its
activity). Dominant negative
mutations generally result in an altered molecular function (often inactive).
For example, the
present disclosure provides dominant negative variants of MMR proteins that
inhibit the activity
of wild-type MMR proteins (e.g., the dominant negative MLH1 proteins described
herein).
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Edit template
[182] The term "edit template" refers to a portion of the extension arm that
encodes the desired
edit in the single strand 3' DNA flap that is synthesized by the polymerase,
e.g., a DNA-
dependent DNA polymerase, RNA-dependent DNA polymerase (e.g., a reverse
transcriptase).
Certain embodiments described here refer to "an RT template," which refers to
both the edit
template and the homology arm together, i.e., the sequence of the PEgRNA
extension arm which
is actually used as a template during DNA synthesis. The term "RT edit
template" is also
equivalent to the term "DNA synthesis template," but wherein the RT edit
template reflects the
use of a prime editor having a polymerase that is a reverse transcriptase, and
wherein the DNA
synthesis template reflects more broadly the use of a prime editor having any
polymerase.
Extension arm
[183] The term "extension arm" refers to a nucleotide sequence component of a
PEgRNA
which comprises a primer binding site and a DNA synthesis template (e.g., an
edit template and
a homology arm) for a polymerase (e.g., a reverse transcriptase). In some
embodiments, the
extension arm is located at the 3' end of the guide RNA. In other embodiments,
the extension
arm is located at the 5' end of the guide RNA. In some embodiments, the
extension arm
comprises a DNA synthesis template and a primer binding site. In some
embodiments, the
extension arm comprises the following components in a 5' to 3' direction: the
DNA synthesis
template and the primer binding site. In some embodiments, the extension arm
also includes a
homology arm. In various embodiments, the extension arm comprises the
following components
in a 5' to 3' direction: the homology arm, the edit template, and the primer
binding site. Since
polymerization activity of the reverse transcriptase is in the 5' to 3'
direction, the preferred
arrangement of the homology arm, edit template, and primer binding site is in
the 5' to 3'
direction such that the reverse transcriptase, once primed by an annealed
primer sequence,
polymerizes a single strand of DNA using the edit template as a complementary
template strand.
[184] The extension arm may also be described as comprising generally two
regions: a primer
binding site (PBS) and a DNA synthesis template, for instance. The primer
binding site binds to
the primer sequence that is formed from the endogenous DNA strand of the
target site when it
becomes nicked by the prime editor complex, thereby exposing a 3' end on the
endogenous
nicked strand. As explained herein, the binding of the primer sequence to the
primer binding site
on the extension arm of the PEgRNA creates a duplex region with an exposed 3'
end (i.e., the 3'
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of the primer sequence), which then provides a substrate for a polymerase to
begin polymerizing
a single strand of DNA from the exposed 3' end along the length of the DNA
synthesis template.
The sequence of the single strand DNA product is the complement of the DNA
synthesis
template. Polymerization continues towards the 5' of the DNA synthesis
template (or extension
arm) until polymerization terminates. Thus, the DNA synthesis template
represents the portion of
the extension arm that is encoded into a single strand DNA product (i.e., the
3' single strand
DNA flap containing the desired genetic edit information) by the polymerase of
the prime editor
complex and which ultimately replaces the corresponding endogenous DNA strand
of the target
site that sits immediately downstream of the PE-induced nick site. Without
being bound by
theory, polymerization of the DNA synthesis template continues towards the 5'
end of the
extension arm until a termination event. Polymerization may terminate in a
variety of ways,
including, but not limited to (a) reaching a 5' terminus of the PEgRNA (e.g.,
in the case of the 5'
extension arm wherein the DNA polymerase simply runs out of template), (b)
reaching an
impassable RNA secondary structure (e.g., hairpin or stem/loop), or (c)
reaching a replication
termination signal, e.g., a specific nucleotide sequence that blocks or
inhibits the polymerase, or
a nucleic acid topological signal, such as, supercoiled DNA or RNA.
Fusion protein
11851 The term "fusion protein" as used herein refers to a hybrid polypeptide
which comprises
protein domains from at least two different proteins. One protein may be
located at the amino-
terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal
(C-terminal)
protein thus forming an "amino-terminal fusion protein" or a "carboxy-terminal
fusion protein,"
respectively. A protein may comprise different domains, for example, a nucleic
acid binding
domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the
protein to a target
site) and a nucleic acid cleavage domain or a catalytic domain of a nucleic-
acid editing protein.
Another example includes a Cas9 or equivalent thereof to a reverse
transcriptase. Any of the
proteins provided herein may be produced by any method known in the art. For
example, the
proteins provided herein may be produced via recombinant protein expression
and purification,
which is especially suited for fusion proteins comprising a peptide linker.
Methods for
recombinant protein expression and purification are well known, and include
those described by
Green and Sambrook, Molecular Cloning: A Laboraloiy Manual (4th ed., Cold
Spring Harbor
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Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of
which are
incorporated herein by reference.
Guide RNA ("gRNA")
[186] As used herein, the term "guide RNA" is a particular type of guide
nucleic acid which is
mostly commonly associated with a Cas protein of a CRISPR-Cas9 and which
associates with
Cas9, directing the Cas9 protein to a specific sequence in a DNA molecule that
includes
complementarity to the protospacer sequence of the guide RNA. However, this
term also
embraces the equivalent guide nucleic acid molecules that associate with Cas9
equivalents,
homologs, orthologs, or paralogs, whether naturally occurring or non-naturally
occurring (e.g.,
engineered or recombinant), and which otherwise program the Cas9 equivalent to
localize to a
specific target nucleotide sequence. The Cas9 equivalents may include other
napDNAbp from
any type of CRISPR system (e.g., type II, V, VI), including Cpfl (a type-V
CRISPR-Cas
systems), C2c1 (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas
system) and C2c3
(a type V CRISPR-Cas system). Further Cas-equivalents are described in
Makarova et al., "C2c2
is a single-component programmable RNA-guided RNA-targeting CRISPR effector,"
Science
2016; 353(6299), the contents of which are incorporated herein by reference.
Exemplary
sequences are and structures of guide RNAs are provided herein. In addition,
methods for
designing appropriate guide RNA sequences are provided herein. As used herein,
the "guide
RNA" may also be referred to as a "traditional guide RNA" to contrast it with
the modified
forms of guide RNA termed "prime editing guide RNAs" (or "PEgRNAs").
[187] Guide RNAs or PEgRNAs may comprise various structural elements that
include, but are
not limited to:
11881 Spacer sequence ¨ the sequence in the guide RNA or PEgRNA (having about
20 nts in
length) which binds to the protospacer in the target DNA.
[189] gRNA core (or gRNA scaffold or backbone sequence) - refers to the
sequence within the
gRNA that is responsible for Cas9 binding, it does not include the 20 bp
spacer/targeting
sequence that is used to guide Cas9 to target DNA. In some embodiments, the
gRNA core or
scaffold comprises a sequence that comprises one or more nucleotide
alterations compared to a
naturally occurring CRISPR-Cas guide RNA scaffold, for example, a Cas9 guide
RNA scaffold.
In some embodiments, the sequence of the gRNA core is designed to comprise
minimal or no
sequence homology to the endogenous sequence of the target nucleic acid at the
target site,
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thereby reducing unintended edits. For example, in some embodiments, one or
more base pairs in
the second stem loop of a Cas9 gRNA core may be "flipped" (e.g., the G-U base
pair and the U-
A base pair as exemplified in Fig. 49A) to reduce unintended edits. In some
embodiments, the
gRNA core comprises no more than 1%, 5%, 10%, 15%, 20%, 25%, or 30% sequence
homology
to the sequence of the double stranded target DNA that flanks 5, 10, 15, 20,
25, 30, 35, 40, 45, or
50 nucleotides upstream or downstream of the position of the one or more
nucleotide edits
[1901 Extension arm - a single strand extension at the 3' end or the 5' end of
the PEgRNA
which comprises a primer binding site and a DNA synthesis template sequence
that encodes via
a polymerase (e.g., a reverse transcriptase) a single stranded DNA flap
containing the genetic
change of interest, which then integrates into the endogenous DNA by replacing
the
corresponding endogenous strand, thereby installing the desired genetic
change.
[191] Transcription terminator - the guide RNA or PEgRNA may comprise a
transcriptional
termination sequence at the 3' of the molecule. in some embodiments, the
PEgRNA comprises a
transcriptional termination sequence between the DNA synthesis template and
the gRNA core.
Homology
[192] The terms "homologous," "homology," or "percent homology" as used herein
refer to the
degree of sequence identity between an amino acid or polynucleotide sequence
and a
corresponding reference sequence. "Homology" can refer to polymeric sequences,
e.g.,
polypeptide or DNA sequences that are similar. Homology can mean, for example,
nucleic acid
sequences with at least about: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%,
83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100%
identity. In other embodiments, a "homologous sequence" of nucleic acid
sequences may exhibit
93%, 95%, or 98% sequence identity to the reference nucleic acid sequence. For
example, a
"region of homology to a genomic region" can be a region of DNA that has a
similar sequence to
a given genomic region in the genome. A region of homology can be of any
length that is
sufficient to promote binding of a spacer or protospacer sequence to the
genomic region. For
example, the region of homology can comprise at least 5, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 200, 300, 400,
500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,
1900, 2000,
2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, or more
bases in length such
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that the region of homology has sufficient homology to undergo binding with
the corresponding
genomic region.
[193] When a percentage of sequence homology or identity is specified, in the
context of two
nucleic acid sequences or two polypeptide sequences, the percentage of
homology or identity
generally refers to the alignment of two or more sequences across a portion of
their length when
compared and aligned for maximum correspondence. When a position in the
compared sequence
can be occupied by the same base or amino acid, then the molecules can be
homologous at that
position. Unless stated otherwise, sequence homology or identity is assessed
over the specified
length of the nucleic acid, polypeptide, or portion thereof. In some
embodiments, the homology
or identity is assessed over a functional portion or a specified portion of
the length.
[194] Alignment of sequences for assessment of sequence homology can be
conducted by
algorithms known in the art, such as the Basic Local Alignment Search Tool
(BLAST)
algorithm, which is described in Altschul eta!, J. Mot Biol. 215:403- 410,
1990. A publicly
available, intemet interface, for performing BLAST analyses is accessible
through the National
Center for Biotechnology Information. Additional known algorithms include
those published in:
Smith & Waterman, "Comparison of Biosequences", Adv. App!. Math. 2:482, 1981;
Needleman
& Wunsch, "A general method applicable to the search for similarities in the
amino acid
sequence of two proteins" J. MoL Biol. 48:443, 1970; Pearson & Lipman
"Improved tools for
biological sequence comparison", Proc. Natl. Acad. Sci. USA 85:2444, 1988; or
by automated
implementation of these or similar algorithms. Global alignment programs may
also be used to
align similar sequences of roughly equal size. Examples of global alignment
programs include
NEEDLE (available at www.ebi.ac.uk/Too1s/psa/emboss_needle0 which is part of
the EMBOSS
package (Rice P etal., Trends Genet., 2000; 16: 276-277), and the GGSEARCH
program
fasta.bioch.virginia.edu/fasta_www2/, which is part of the FASTA package
(Pearson W and
Lipman D, 1988, Proc. Natl. Acad. ScL USA, 85: 2444-2448). Both of these
programs are based
on the Needleman-Wunsch algorithm, which is used to find the optimum alignment
(including
gaps) of two sequences along their entire length. A detailed discussion of
sequence analysis can
also be found in Unit 19.3 of Ausubel eta! ("Current Protocols in Molecular
Biology" John
Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).
[195] A skilled person understands that amino acid (or nucleotide) positions
may be determined
in homologous sequences based on alignment. For example, "H840" in a reference
Cas9
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sequence may correspond to 11839, or another corresponding position in a Cas9
homolog when
aligned to the reference Cas9 sequence.
Host cell
11961 The term "host cell," as used herein, refers to a cell that can host,
replicate, and express a
vector described herein, e.g., a vector comprising a nucleic acid molecule
encoding an MLH1
variant and a fusion protein comprising a Cas9 or Cas9 equivalent and a
reverse transcriptase.
Inhibit
1197] As used herein the term "inhibiting," "inhibit," or "inhibition" in the
context of proteins
and enzymes, for example, in the context of enzymes involved in the DNA
mismatch repair
pathway, refers to a reduction in the activity of the protein or enzyme. In
some embodiments, the
term refers to a reduction of the level of enzyme activity, e.g., the activity
of one or more
enzymes in the DNA mismatch repair pathway, to a level that is statistically
significantly lower
than an initial level, which may, for example, be a baseline level of enzyme
activity. In some
embodiments, the term refers to a reduction of the level of enzyme activity,
e.g., the activity of
one or more enzymes in the DNA mismatch repair pathway, to a level that is
less than 75%, less
than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less
than 10%, less than
9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less
than 3%, less than
2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, less than
0.001%, or less than
0.0001% of an initial level, which may, for example, be a baseline level of
enzyme activity.
Linker
[198] The term "linker," as used herein, refers to a molecule linking two
other molecules or
moieties. The linker can be an amino acid sequence in the case of a linker
joining two fusion
proteins. For example, a Cas9 can be fused to a reverse transcriptase by an
amino acid linker
sequence. The linker can also be a nucleotide sequence in the case of joining
two nucleotide
sequences together. For example, in the instant case, the traditional guide
RNA is linked via a
spacer or linker nucleotide sequence to the RNA extension of a prime editing
guide RNA which
may comprise a RT template sequence and an RT primer binding site. In other
embodiments, the
linker is an organic molecule, group, polymer, or chemical moiety. In some
embodiments, the
linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10,11, 12,
13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-
60, 60-70, 70-80,
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80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter
linkers are also
contemplated. In certain embodiments, the linker is a self-hydrolyzing linker
(e.g., a 2A self-
cleaving peptide as described further herein). Self-hydrolyzing linkers such
as 2A self-cleaving
peptides are capable of inducing ribosomal skipping during protein
translation, resulting in the
ribosome failing to make a peptide bond between two genes, or gene fragments.
MLH1
[199] The term "MLH1" refers to a gene encoding MLH1 (or MutL Homolog 1), a
DNA
mismatch repair enzyme. The protein encoded by this gene can heterodimerize
with mismatch
repair endonuclease PMS2 to form MutL alpha (MutLa), part of the DNA mismatch
repair
system. MLHI mediates protein-protein interactions during mismatch
recognition, strand
discrimination, and strand removal. In mismatch repair, the heterodimer
MSH2:MSH6 (MutSa)
forms and binds the mismatch. MLH1 then forms a heterodimer with PMS2 (MutLa)
and binds
the MSH2:MSH6 heterodimer. The MutLa heterodimer then incises the nicked
strand 5' and 3'
of the mismatch, followed by excision of the mismatch from MutLa-generated
nicks by EX01.
Finally, POLS resynthesizes the excised strand, followed by LIG1
[200] An exemplary amino acid sequence of MLH1 is human isoform 1, P40692-1:
>sp1P406921MLH1JILIMAN DNA mismatch repair protein Mlhl OS=Homo sapiens 0X-
9606
GN=MLH1 PE=1 SV=1:
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLICLIQIQDNGTGIRKEDL
DIVCERPITSKLQSFEDLASIS'FYGFRGEALASISHVAHVTITIKTADGKCAYRASYSDGKLKAPPKPCAGN
QGTQFTVEDLFYNIATRRKALKNPSEEYGKILEVVGRY SVHNAGISFS VKKQGETVADVR'FLPNASTVDNIR
SIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVICKCIFLLFINHRLVESTSLRKAIETVYAKYLPKNTHPF
LYLSLEISPQNVDVNVIIPTICHEVHFLHEESILERVQQMESKLLGSNSSRMYFTQTLLPGLAGPSGEMVKST
TSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSICPLSSQPQAIVTEDKTDISSGRARQQDEEMLELP
APAEVAAICNQSLEGDITICGTSEMSEKRGPTSSNPRICRHREDSDVEMVEDDSRKEMTAACTPRRRIINLTSV
LSWEEINEQGHEVLREIVILIINHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLS
EPAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLICKKAEMLADYF'SLEIDEEGNLIGLPLUDNYVPPL
EGLPIFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKA
LRSHILPPKIIF'rEDGNILQLANLPDLYICVFERC (SEQ ID NO: 204),
or an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
up to and including
100% sequence identity with SEQ ID NO: 204.
[201] Another exemplary amino acid sequence of MLH1 is human isoform 2, P40692-
2
(wherein amino acids 1-241 of isoform 1 are missing): >spIP40692-21MLH1_HUMAN
lllsofonn 2
of DNA mismatch repair protein Mlhl OS=Homo sapiens 0X=9606 GN=MLH1:
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MNGY I SNANYSVKKCIFT.LFINITRINESTSLIZ K AIETVYA AYLPKNTE PF SLEISPQNVDVNVI-I
PTKITE
VHFLHEESILERVQQHIESKLLG SNS SRMYFTQTLLPGLAGPSGEM VK S TTSLTSSSTSGSSDKVYAHQMVR
TDSREQKLDAFLQPISKPL S SQPQAWTEDK.TDI SSGRARQQDEEMLEIPAP AEVAAKNQSLEGDTTKGTSE
MSEKRGPTSSNPRKRHRED SDVEMVEDDSRKEMTAACTPRRRIINLTSVL SLQEEINEQGHEVLREMLHNH
SFVGCVNPQWALAQHQTKLYLLNTFKLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTE
EDGPKEGLAEYIVEFLKKKAEML ADYFSLEIDEEGNLIGLPILIDNY VPPLEGLPIFILRLATEVNWDEEKEC
FESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWIVEHIVYKAIRSHILPPICHF1 __ EUGNILQL
AN
LPDLYKVFERC (SEQ ID NO: 205),
or an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
up to and including
1000/0 sequence identity with SEQ ID NO: 205.
12021 Another exemplary amino acid sequence of MLH1 is human isoform 3, P40692-
3 (where
amino acids 1-101 (MSFVAGVIRR...ASISTYGFRG (SEQ ID NO: 206) is replaced with
MAF): >spIP40692-2[MLH1 HUMAN Isoform 2 of DNA mismatch repair protein Mlhl
OS=Homo sapiens OX=9606 GN=MLH1:
MAFEALASISHVAHVTITTKTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKALKNP
SEEYGKILEVVGRYSVHNAGI SF SVKKQGETVADVRTLPNASTVDNIRSIFGNAVSRELIEIGCEDKTL AFKM
NGYISNANYSVICKCIFLLFINIIRLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISPQNVDVNVIIPTKIIEVH
FLIMPSILERVQQIIIESKLLGSNSSRMYFTQTLLPGLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRTD
SREQICLDAFLQPLSKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVAAICNQSLEGDTTKOTSEMS
EKROPTSSNPRKRHREDSDVEMVEDDSRKEMTAACTPRRRIINLTSVLSLQEEINEQGHEVLREMLIINHSF
VGCVNPQWALAQHQTKLYLLNTI7CLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEED
GPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFII,RLATEVNWDEEKECFES
LSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKAIRSHILPPKIIFTEDGNILQLANLPD
LYKVFERC (SEQ ID NO: 207),
or an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
up to and including
100% sequence identity with SEQ ID NO: 207.
[203] The present disclosure contemplates targeting MLH1 and/or MMR pathway
components
that interact with MLHI, including any wildtype or naturally occurring variant
of MLHI,
including any amino acid sequence having at least 70%, or 75%, or 80%, or 85%,
or 90%, or
95%, or 99% or more sequence identity with any of SEQ ID NOs: 204, 208-213,
215, 216, 218,
222, or 223, or nucleic acid molecules encoding any MLH1 or variant of MLH1
(e.g., a
dominant negative mutant of MLHI as described herein), for inhibiting,
blocking, or otherwise
inactivating the wild type MLH1 function in the MMR pathway, and consequently,
inhibiting,
blocking, or otherwise inactivating the MMR pathway, e.g., during genome
editing with a prime
editor.
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[204] In some embodiments, inactivation of the MMR pathway involves an
inhibitor that
disrupts, blocks, interferes with, or otherwise inactivates the wild type
function of the MLH1
protein. In some embodiments, inactivation of the MMR pathway involves a
mutant of the
MLH1 protein, for example, contacting a target cell with a MLH1 mutant protein
or expressing
in a target cell an MLH1 mutant nucleic acid that encodes an MLH1 mutant
protein. In some
embodiments, the MLH1 mutant protein interferes with, and thereby inactivates,
the function of
a wild type MLH1 protein in the MMR pathway. In some embodiments, the MLH1
mutant is a
dominant negative mutant. In some embodiments, the MLH mutant protein is
capable of binding
to an MLH1-interacting protein, for example, MutS.
[205] Without being bound by theory, MLH1 dominant negative mutants function
by saturating
binding of MutS, thereby blocking MutS-wild type MLH1 binding and interfering
with the
function of the wild type MLH1 protein in the MMR pathway.
[206] In various embodiments, the dominant negative IMLHI can include, for
example, MLH1
E34A, which is based on SEQ ID NO: 222 and having the following amino acid
sequence
(underline and bolded to show the E34A mutation):
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIICAMIENCLDAKSTSIQVIVKEGGLKLIQIQDNGTGIRKEDL
DIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTADGKCAYRASYSDGKLICAPPKPCAGN
QGTQITVEDLFYNIATRRKALKNPSEEYGKILEVVGRYSVHNAGISFSVKICQGETVADVRTLPNASTVDNIR
SIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRICAIETVYAAYLPKNTHPF
LYLSLEISPQNVIWNVHPTICHEVHFLHEESILERVQQ141ESKLLGSNSSRMYFTQTLLPGLAGPSGEMVKST
TSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELP
APAEVAAKNQSLEGDTIKGTSEMSEKRGPTSSNPRKRHREDSDVEMVEDDSRICEMTAACT'PRRREINLTSV
LSLQEEINEQGIIEVLREMLHNHSFVGCVNPQWALAQHQTKLYLLNITKLSEELFYQILIYDFANFGVLRLS
EPAPLFDLAINALALDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPL
EGLPIFILRL ATE VNWDEEXECFESLSKECAMFY SIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKA
IASHILPPICHFTEDGNILQLANLPDLYKVFERC (SEQ ID NO: 222),
or an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least
900/o, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or up to and including
100% sequence identity with SEQ ID NO: 222.
12071 In various other embodiments, the dominant negative MLH1 can include,
for example,
MLH1 A756, which is based on SEQ ID NO: 208 and having the following amino
acid sequence
(underline and bolded to show the A756 mutation at the C terminus of the
sequence):
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAMEMIENCLDAKSTSIQVIVKEGGLICLIQIQDNOTGIRKEDL
DIVCERFITSKLQSFEDLASISTYGFRGEALASISHVAHVTITTICTADGKCAYRASYSDGKLKAPPICPCAGN
QGTQITVEDLFYNIATRRICALKNPSEEYGICILEVVGRYSVIINAGISFSVKKQGETVADVRTLPNASTVDN1R
SUFGNAVSRELIEIGCEDICILAFKNINGYISNANYSVKKCIFLLFINIIRLVESTSLIMMETVYAAYLPKNTIIPF
LYLSLEISPQNVDVNVIIPTKREVHFLIIEESILERVQQMESKLLGSNSSRMYFTQTLLPGLAGPSGEMVKST
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TSLTSSSTSGSSDKVYAHQMVRTDSREQKID AFLQPLSKPLSSQPQAIVT.EDKTDISSGRARQQDEEML ELY
APAEVAAKNQSLEGDITKGTSEMSEKRGPTSSNPRICRHREDSDVEMVEDDSRKEMTAACTPRRRIINLTSV
LSLQEEINEQGHEVLREML:HNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFG'VLRLS
EPAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLKICKAEMLADYFSLEIDMGNLIGLPLLIDNYVPPL
EGLI:VITAL ATEVNVVDEFICECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKA.
LRSHILPPKHF1EDGNILQLANLPDLYKVFERH(SEQ ID NO: 208),
or an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
up to and including
100% sequence identity with SEQ ID NO: 208 (wherein the [-] indicates deleted
amino acid
residue(s) relative to the parent or wildtype sequence).
12081 In still other embodiments, the dominant negative MLH1 can include, for
example,
MLH1 A754-A756, which is based on SEQ ID NO: 209 and having the following
amino acid
sequence (underline and bolded to show the A754-A756 mutation at the C
terminus of the
sequence):
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCIDAKSTSIQVIVKEGGLICLIQIQDNGTGIIRKEDL
DIVCERFITSKLQSFE.DLASISTYGFRGEALASISHVAHVTITTKTADGXCAYRASYSDGKLKAPPICPCAGN
QGTQITVEDLEYNIATRRKALKNPSEEYGKILEVVGRYSVHNAGISFSVICKQGETVADVRTLPNASTVDNIR
SIFGNAVSRELIEIGC.EDKTLAFKMNGYISNANYS'VKKCIFLLFINHRLVESTSLRKAIETVY AAYLPKNTHPF
LYLSLEISPQNV.DVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQTLLPGLAGPSG.EMVKST
TSLTSSSTSGSSDKVYAHQMVR.TDSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELP
APAEVAAKNQSLEG.DTT.KGTSEMSEKRGPTSSNPRKRHREDSDVEMV.EDDSRKEMTAACT.PRRRIINLTSV
LSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQT.KLYLLN'TTKLSEELFYQILIYDFANFGVLRLS
EPAPLFDLAMLALDSPESGWTEEDGPICEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPL
EGLPIFILRLATEVNWDEEKECFESLSICECAMFYS1RKQYISEESTLSGQQSEVPGSIPNSWK.WTVEHIVYKA
LRSHILPPKHFTEDGNILQLANLPDLYKVFF - (SEQ ID NO: 209),
or an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
up to and including
100% sequence identity with SEQ ID NO: 209 (wherein the [- - -] indicates
deleted amino acid
residue(s) relative to the parent or wildtype sequence).
12091 In yet other embodiments, the dominant negative MLH1 can include, for
example, MUD
E34A A754-A756, which is based on SEQ ID NO: 210 and having the following
amino acid
sequence (underline and bolded to show the E34A and A754-A756 mutations):
MSFVAGVIRRLDETVVNRIAAGE'VIQRPANAIKAMIENCLDAKSTSIQVIVKEGGLKLIQIQDNGTGIRICEDL
DIVCMFTTSKLQSFEDLASISTYGFRGEALASISHVAHV'ITITKTADGKCAYRASYSDGKLKAPPKF'CAGN
QGTQITVEDLFYNIATRRKALKNPSEEYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIR
SIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPF
LYLSLEISPQNVDVNVIIPTKHEVHFLHEESILERVQQHTESKLLGSNSSRMYFTQTLLPGLAGPSGEMVKST
TSLTSSSTSGSSDKVYAHQMVR'FDSRF,QKLDAFLQPLSKPLSSQPQAIVTEDKTDISSGRAR.QQDEEMLELP
APAEVAAICNQSLEGDTIXGTSEMSEKRGPTSSNPRKRHREDSDVE'MVEDDSRKEMTAACTPRRMINLTSV
LSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLS
EPAP.LFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLTDNYVPPL
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EGLPIF II,RI, ATE VNWDEEK
ECFESLSKECAMFYS1RKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKA.
IRSHILPPIGIFTEDGNILQLANLPDLYKVF[- - -] (SEQ ID NO: 210),
or an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
up to and including
100% sequence identity with SEQ ID NO: 210.
12101 In certain embodiments, the dominant negative MIAll can include, for
example, :MLH1
1-335, which is based on SEQ ID NO: 211 and having the following amino acid
sequence
(contains amino acids 1-335 of SEQ NO: 204):
MSFVAGVIRRLDETVVN.R1AAGEVIQRPANAIKEMIENCLDAKSTS1QVIVKEGGLKLIQIQDNGTGIRKEDL
DIVCERFTTSKLQSPEDLASISTYGFR.GEALASISHVAHVTITTKTADGKCAYRASYSDGKLKAPPKPCAGN
QGTQITVEDLFYNIATRRKALKNPSEEYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDN1R
SIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVICKCIFLLFINHRLVESTSLRICAIETVYAAYLPKNTHPF
LYLSLEISPQNVDVNVHPTKHEVHFLHEES1LERVQQHIESKLL (SEQ ID NO: 211),
or an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
up to and including
100% sequence identity with SEQ 1E) NO: 211.
[211] In other embodiments, the dominant negative MLH1 can include, for
example, MLH1 1-
335 E34A, which is based on SEQ 113 NO: 212 and having the following amino
acid sequence
(contains amino acids 1-335 of SEQ NO: 204 and a E34A mutation relative to SEQ
ID NO:
204):
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKAMIENCLDAKSTSIQVIVICEGGLKLIQIQDNGTOIRICEDL
DIVCERFITSKLQSFEDLASISTYGFRGEALASISHVAHVTITIKTADGKCAYRASYSDGKIKAPPICPC.AGN
QGTQITVEDLFYNIA.TRRKALKNPSEEYGKILEVVGRYSVIINAGISFSVKICQGETVAD'VRTI,PNA.STVDNIR

SIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYS'VKICCIFILFINFIRINESTSIRKAIETVYAAYLPKNTHPF
LYI-SLEISPQNVDVNVHPTIMEVHFIEFESILERVQQHIESKLL (SEQ ID NO: 212),
or an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
up to and including
100% sequence identity with SEQ ID NO: 212.
[212] In still other embodiments, the dominant negative MLH1 can include, for
example,
MLH1 1-335 NLSs'm (or referred to as MLH1CITINT0, which is based on SEQ ID NO:
204 and
having the following amino acid sequence (contains amino acids 1-335 of SEQ
NO: 204 and an
NLS sequence of SV40):
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKENIIENCLDAKSTSIQVIVKEGGLKLIQIQDNGTGIRKEDL
DIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVIITTK'FADGKCAYRASYSDGKLICAPPKPCAGN
QGTQITVEDLFYNIATRRKALKNPSEEYGICILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIR
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SIFGNAVSRELIEIGCEDKTI.AFKMNGYISNANYSVKKCIFLLFINIIRINESTSLRKATETVYAAYLPKNTHPF
LYLSLEISPQNVDVNVI-IPTKHEVHFIBEESILERVQQHIESKLLPIKKKRKV (SEQ NO: 213),
with the underlined and bolded portion referring to the NLS of SV40), or an
amino acid
sequence having at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or up to and including
100% sequence
identity with SEQ ID NO: 213.
[2131 In still other embodiments, the dominant negative MLH1 can include, for
example,
MLH I 1-335 NLSaliernate (which is based on SEQ ID NO: 204 and having the
following amino
acid sequence (contains amino acids 1-335 of SEQ NO: 204 and an alternate NLS
sequence)):
MSFVAGVIRRLDETVNINR1AAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLKLIQIQDNGTGIRKEDL
DIVCERFITSICLQSFEDLASISTYGFRGEALASISHVAHVIIITKTADGKCAYRASYSDGKLICAPPKPCAGN
QGTQITVEDLFYNIATRRICALKNPSEEYGICILEVVGRYSVIINAGISFSVKKQGETVAD'VRTLPNASTVDNIR
SIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINIIRLVESTSLRKAIETVYAAYLPICN'TIIPF

LYLSLEISPQNVDVNVHPTKHEVHFLBFFSILERVQQHIESKLL-[alternate NLS sequence] (SEQ ID
NO: 214)-
[alternate NLS sequence],
or an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
up to and including
100% sequence identity with SEQ ID NO: 214. The alternate NLS sequence can be
any suitable
NLS sequence, including but not limited to:
Description Sequence SEQ ID NO:
NLS MKRTADGSEFESPKKKRKV SEQ ID NO: 101
NLS MDSLLMNRRKFLYQFICNVRWAKGRRETYLC SEQ ID NO: 1
NLS of nucleoplasmin A VKIIPAATKKAGQAKKKKID SEQ ID NO: 133
NLS of EGL-13 MSRRRICANPTKLSENAKKLAICEVEN SEQ ID NO: 134
NLS of c-MYC PAAKRVKLD SEQ ID NO: 135
NLS of TUS-protein KLKIKRPVK SEQ ID NO: 136
NLS of polyoma large T-Ag VSRKRPRP SEQ
ID NO: 137
NLS of Hepatitis r) virus antigen EGAPPAKRAR SEQ ID NO: 138
NLS of murine p53 PPQPKKKPLDGE SEQ ID NO: 139
NLS of PEI and PE2 SGGSKRTADGSEFhPKKKRKV SEQ ID NO: 103
In some embodiments, an NLS sequence is appended to the N-terminus of a
protein and begins
with a methionine ("M"). In other embodiments, an NLS sequence may be appended
at the C-
terminus of a protein, or between multiple domains of a fusion protein, and
does not begin with a
methionine (i.e., the M in, for example, SEQ ID NOs: 101, 1, and 134 is not
included in the NLS
when it is appended at the C-terminus or between two domains in a fusion
protein).
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[214] In still other embodiments, the dominant negative MLII1 can include, for
example,
MLH1 501-756, which corresponds to a C-terminal fragment of SEQ ID NO: 204
that
corresponds to amino acids 501-756 of SEQ ID NO: 204:
INLTSVLSLQEEINEQGHEVLRENILHNHSFVGCVNPQWALAQHQTKLYLLNTTICI,SF,ELFYQII,IYDFANFG
VLRLSEPAPLFDLAMLALDSPESGWTEEDGPKF,GIAEYIVEFLKKICAEMLADYFSLEIDEEGNLIGLPLIADN
YVPPLEGLPIFILRIATEVNWDEFICECFESLSKECAMFYSIRKQYISEESTI,SGQQSEVPGSIPNSWKWTVEHI
VYKALRSHILPPKHFTF,DGNR,QLANI,PDLYK.VFF,RC (SEQ ID NO: 215),
or an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
up to and including
100% sequence identity with SEQ NO: 215.
[215] in still other embodiments, the dominant negative MLH1 can include, for
example,
MLH1 501-753, which corresponds to a C-terminal fragment of SEQ ID NO: 204
that
corresponds to amino acids 501-753 of SEQ ID NO: 204:
INLTSVLSWEEINEQGHEVLREMIENHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQ11,IYDFANFG
VLRLSEPAPLFDLAMLAIDSPESGWTEEDGPKEGLAEYIVEFLICKKAEMLADYFSLEIDEEGNLIGLPILIDN
YVPPLEGLPIFTLRIATEVNWDEEKECI. __ ESL SKECAMFYSERKQYISEES71,
SGQ9SEVPGSIPNSWKWTVFIII
VYKALRSHILPPKIIFFEDGNILQLANLPDLYK.VFF - (SEQ ID NO: 216),
or an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
up to and including
100% sequence identity with SEQ ID NO: 216.
[216] In still other embodiments, the dominant negative MLEI1 can include, for
example,
MLH1 461-756, which is a C-terminal fragment of SEQ ID NO: 204 that
corresponds to amino
acids 461-756 of SEQ ID NO: 204:
KRGPTSSNPRKRHREDSDVFNIVEDDSRKEMTAACTPRRRIINLTSVISI.,QEEINF-QGBEVLREMIRNHSFV
GCVNPQWALAQHQTKLYLLNITKI,SEELFYQ11,1YDFANFGVLRLSEPAPLFDLAMLALDSPESGWTF.EDG
PKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPI.LIDNYVPPLEGLPIFILRLATEVNWDEEKECFESL
SKECANIFYSIRKQYISEESTI,SGQQSEVPGSIPNSWKWTVEHIVYKAIRSHILPPKHFIEDGNILQI,ANI,PDL,

YKVFERC (SEQ ID NO: 217),
or an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
up to and including
100% sequence identity with SEQ B3 NO: 217.
[217] In various embodiments, the dominant negative MLH1 can. include, for
example. MLII1
461-753, which is a C-terminal fragment of SEQ ID NO: 204 that corresponds to
amino acids
461-753 of SEQ ID NO: 204:
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KRGPTSSNPRICRHREDSDVFIvIVEDDSRKEMTAACTPR RRIINLTS SLQEEINEQGHEVI,REMLIINHSFV
GCVNPQWALAQHQTKLYI,I,NITKLSEET,FYQ11.1YDFANFGVLRI,SF,PAPLFDLAMLAI,DSPESGWTEEDG

PKEGLAEYI'VEFLKKKAEMLADYFSLEIDEEGNLIGLPLUDNYVPPLEGLPIPTIALATEVNWDEXKECFESI,
SKECANIFYSIRKQYISEESTLSGQQSE'VPGSIPNSWKWTVEHTVYKALRSHILPPKHF _________________
I EDGNILQLANI.PDI,
YKVF[- - -] (SEQ ID NO: 218),
or an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
up to and including
100% sequence identity with SEQ ID NO: 218.
In various other embodiments, the dominant negative MLH1 can include, for
example, MLH1
461-753, which is a C-terminal fragment of SEQ ID NO: 204 that corresponds to
amino acids
461-753 of SEQ ID NO: 204, and which further comprises an N-terminalNLS, e.g.,
NLSsv :
[NLSI-
KRGPTSSNPRKRHREDSDVEMVEDDSRKEMTAACIPRRRIINLTSVLSLQEEINEQGHEVLREMLHNHSFV
GCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFOVLRLSEPAPLFDLAMLALDSPESGWTEEDG
PKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESL
SKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKALRSHILPPICHFTEDGN1LQLANLPDL
YKVF[- - -1 (SEQ ID NO: 218),
or an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
up to and including
100% sequence identity with SEQ ID NO: 218. The NLS sequence can be any
suitable NLS
sequence, including but not limited to SEQ ID NOs: 1, 101, 103, 133-139.
napDNAbp
12181 As used herein, the term "nucleic acid programmable DNA binding protein"
or
"napDNAbp," of which Cas9 is an example, refer to proteins that use RNA:DNA
hybridization
to target and bind to specific sequences in a DNA molecule. Each napDNAbp is
associated with
at least one guide nucleic acid (e.g., vide RNA), which localizes the napDNAbp
to a DNA
sequence that comprises a DNA strand (i.e., a target strand) that is
complementary to the guide
nucleic acid, or a portion thereof (e.g., the protospacer of a guide RNA). In
other words, the
guide nucleic-acid "programs" the napDNAbp (e.g., Cas9 or equivalent) to
localize and bind to a
complementary sequence.
[219] Without being bound by theory, the binding mechanism of a napDNAbp ¨
guide RNA
complex, in general, includes the step of forming an R-loop whereby the
napDNAbp induces the
unwinding of a double-strand DNA target, thereby separating the strands in the
region bound by
the napDNAbp. The guide RNA spacer sequence then hybridizes to the "target
strand." This
displaces a "non-target strand" that is complementary to the target strand,
which forms the single
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strand region of the R-loop. In some embodiments, the napDNAbp includes one or
more
nuclease activities, which then cut the DNA, leaving various types of lesions.
For example, the
napDNAbp may comprises a nuclease activity that cuts the non-target strand at
a first location,
and/or cuts the target strand at a second location. Depending on the nuclease
activity, the target
DNA can be cut to form a "double-stranded break" whereby both strands are cut.
In other
embodiments, the target DNA can be cut at only a single site, i.e., the DNA is
"nicked" on one
strand. Exemplary napDNAbp with different nuclease activities include "Cas9
nickase"
("nCas9") and a deactivated Cas9 having no nuclease activities ("dead Cas9" or
"dCas9").
Exemplary sequences for these and other napDNAbp are provided herein.
Nickase
[220] The term "nickase," as used herein, may refer to a Cas9 with one of the
two nuclease
domains inactivated. This enzyme is capable of cleaving only one strand of a
target DNA. As
used herein, a "nickase" may refer to a napDNAbp (e.g., a Cas protein) which
is capable of
cleaving only one of the two complementary strands of a double-stranded target
DNA sequence,
thereby generating a nick in that strand. In some embodiments, the nickase
cleaves a non-target
strand of a double stranded target DNA sequence. In some embodiments, the
nickase comprises
an amino acid sequence with one or more mutations in a catalytic domain of a
canonical
napDNAbp (e.g., a Cas protein), wherein the one or more mutations reduces or
abolishes
nuclease activity of the catalytic domain. In certain embodiments, the
napDNAbp is a Cas9
nickase, a Cas12a nickase, or a Cas12b1 nickase. In some embodiments, the
nickase is a Cas9
that comprises one or more mutations in a RuvC-like domain relative to a wild
type Cas9
sequence or to an equivalent amino acid position in other Cas9 variants or
Cas9 equivalents. In
some embodiments, the nickase is a Cas9 that comprises one or more mutations
in an HNH-like
domain relative to a wild type Cas9 sequence or to an equivalent amino acid
position in other
Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9
that comprises an
aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of
Cas9 relative to a
canonical Cas9 sequence or to an equivalent amino acid position in other Cas9
variants or Cas9
equivalents. In some embodiments, the nickase is a Cas9 that comprises a
H840A, N854A,
and/or N863A mutation relative to a canonical Cas9 sequence, or to an
equivalent amino acid
position in other Cas9 variants or Cas9 equivalents. In some embodiments, the
term "Cas9
nickase" refers to a Cas9 with one of the two nuclease domains inactivated.
This enzyme is
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capable of cleaving only one strand of a target DNA. In some embodiments, the
nickase is a Cas
protein that is not a Cas9 nickase.
12211 In some embodiments, the napDNAbp of the prime editing complex comprises
an
endonuclease having nucleic acid programmable DNA binding ability. In some
embodiments,
the napDNAbp comprises an active endonuclease capable of cleaving both strands
of a double
stranded target DNA. In some embodiments, the napDNAbp is a nuclease active
endonuclease,
e.g., a nuclease active Cas protein, that can cleave both strands of a double
stranded target DNA
by generating a nick on each strand. For example, a nuclease active Cas
protein can generate a
cleavage (a nick) on each strand of a double stranded target DNA. In some
embodiments, the two
nicks on both strands are staggered nicks, for example, generated by a
napDNAbp comprising a
Cas12a or Cas12b1. In some embodiments, the two nicks on both strands are at
the same
genomic position, for example, generated by a napDNAbp comprising a nuclease
active Cas9. In
some embodiments, the napDNAbp comprises an endonuclease that is a nickase.
For example, in
some embodiments, the napDNAbp comprises an endonuclease comprising one or
more
mutations that reduce nuclease activity of the endonuclease, rendering it a
nickase. In some
embodiments, the napDNAbp comprises an inactive endonuclease. For example, in
some
embodiments, the napDNAbp comprises an endonuclease comprising one or more
mutations that
abolish the nuclease activity. In various embodiments, the napDNAbp is a Cas9
protein or
variant thereof. The napDNAbp can also be a nuclease active Cas9, a nuclease
inactive Cas9
(dCas9), or a Cas9 nickase (nCas9). In a preferred embodiment, the napDNAbp is
Cas9 nickase
(nCas9) that nicks only a single strand. In certain embodiments, the napDNAbp
is a Cas9
nickase, a Cas12a nickase, or a Cas12b1 nickase. In some embodiments, the
napDNAbp can be
selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1,
Cas12b2,
Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Casl2f (Cas14),
Casl2f1, Cas12j
(Case), and Argonaute, and optionally has a nickase activity such that only
one strand is cut. In
some embodiments, the napDNAbp is selected from Cas9, Cas12e, Casl 2d, Casl
2a, Cas12b1,
Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f
(Cas14), Casl2f1,
Casi2j (Case), and Argonaute , and optionally has a nickase activity such that
one DNA strand
is cut preferentially to the other DNA strand.
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Nick site
[222] The terms "cleavage site," "nick site," and "cut site" as used
interchangeably herein in
the context of prime editing, refer to a specific position in between two
nucleotides or two base
pairs in the double-stranded target DNA sequence. lin some embodiments, the
position of a nick
site is determined relative to the position of a specific PAM sequence. In
some embodiments, the
nick site is the particular position where a nick will occur when the double
stranded target DNA
is contacted with a napDNAbp, e.g., a nickase such as a Cas nickase, that
recognizes a specific
PAM sequence. For each PEgRNA described herein, a nick site is characteristic
of the particular
napDNAbp to which the gRNA core of the PEgRNA associates with, and is
characteristic of the
particular PAM required for recognition and function of the napDNAbp. For
example, for a
PEgRNA that comprises a gRNA core that associates with a SpCas9, the nick site
in the
phosphodiester bond between bases three ("-3" position relative to the
position 1 of the PAM
sequence) and four ("-4" position relative to position l of the PAM sequence).
[223] In some embodiments, a nick site is in a target strand of the double-
stranded target DNA
sequence. In some embodiments, a nick site is in a non-target strand of the
double-stranded
target DNA sequence. In some embodiments, the nick site is in a protospacer
sequence. In some
embodiments, the nick site is adjacent to a protospacer sequence. In some
embodiments, a nick
site is downstream of a region, e.g., on a non-target strand, that is
complementary to a primer
binding site of a PEgRNA. In some embodiments, a nick site is downstream of a
region, e.g., on
a non-target strand, that binds to a primer binding site of a PEgRNA. In some
embodiments, a
nick site is immediately downstream of a region, e.g., on a non-target strand,
that is
complementary to a primer binding site of a PEgRNA. In some embodiments, the
nick site is
upstream of a specific PAM sequence on the non-target strand of the double
stranded target
DNA, wherein the PAM sequence is specific for recognition by a napDNAbp that
associates
with the gRNA core of a PEgRNA. In some embodiments, the nick site is
downstream of a
specific PAM sequence on the non-target strand of the double stranded target
DNA. wherein the
PAM sequence is specific for recognition by a napDNAbp that associates with
the gRNA core of
a PEgRNA. In some embodiments, the nick site is 3 nucleotides upstream of the
PAM sequence,
and the PAM sequence is recognized by a Streptococcus pyogenes Cas9 nickase, a
P.
lavamentivorans Cas9 nickase, a C diphtheriae Cas9 nickase, a N. cinerea Cas9,
a S. aureus
Cas9, or a N. lari Cas9 nickase. In some embodiments, the nick site is 3
nucleotides upstream of
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the PAM sequence, and the PAM sequence is recognized by a Cas9 nickase,
wherein the Cas9
nickase comprises a nuclease active HNH domain and a nuclease inactive RuvC
domain. In
some embodiments, the nick site is 2 base pairs upstream of the PAM sequence,
and the PAM
sequence is recognized by a S'. thermophilus Cas9 nickase.
Nucleic acid molecule
[2241 The term "nucleic acid," as used herein, refers to a polymer of
nucleotides. The polymer
may include natural nucleosides (i.e., adenosine, thymidine, guanosine,
cytidine, uridine,
deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside
analogs (e.g.,
2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl
adenosine, 5-
methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyl
uridine, C5
propynyl cytidine, C5 methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8
oxoadenosine, 8
oxoguanosine, 0(6) methylguanine, 4-acetylcytidine, 5-
(carboxyhydroxymethyl)uridine,
dihydrouridine, methylpseudouridine, 1-methyl adenosine, 1-methyl guanosine,
N6-methyl
adenosine, and 2-thiocytidine), chemically modified bases, biologically
modified bases (e.g.,
methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose,
ribose, 2'-
deoxyribose, 2'-0-methylcytidine, arabinose, and hexose), or modified
phosphate groups (e.g.,
phosphorothioates and 5'N phosphoramidite linkages).
P53
12251 As used herein, "p53" refers to tumor protein 53. Among other functions,
p53 plays a role
in DNA damage and repair, specifically in its role in regulation of the cell
cycle, apoptosis, and
genomic stability. P53 can activate DNA repair proteins when DNA has been
damaged. P53 may
also arrest cell growth by holding the cell cycle at the Gl/S regulation point
on DNA damage
recognition, thereby providing DNA repair proteins more time to fix the DNA
damage before
allowing the cell to continue the cell cycle. Thus, in some embodiments of the
methods described
herein, p53 is inhibited (e.g., by the p53 inhibitor protein "i53," or another
p53 inhibitor) to
increase the efficiency of prime editing.
PEgRNA
12261 As used herein, the terms "prime editing guide RNA" or "PEgRNA" or
"extended guide
RNA" refer to a specialized form of a guide RNA that has been modified to
include one or more
additional sequences for implementing the prime editing methods and
compositions described
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herein. As described herein, the prime editing guide RNA comprise one or more
"extended
regions" of nucleic acid sequence. The extended regions may comprise, but are
not limited to,
single-stranded RNA or DNA. Further, the extended regions may occur at the 3'
end of a
traditional guide RNA. In other arrangements, the extended regions may occur
at the 5' end of a
traditional guide RNA. In still other arrangements, the extended region may
occur at an
intramolecular region of the traditional guide RNA, for example, in the gRNA
core region which
associates and/or binds to the napDNAbp. The extended region comprises a "DNA
synthesis
template" which encodes (by the polymerase of the prime editor) a single-
stranded DNA which,
in turn, has been designed to be (a) homologous with the endogenous target DNA
to be edited,
and (b) which comprises at least one desired nucleotide change (e.g., a
transition, a transversion,
a deletion, or an insertion) to be introduced or integrated into the
endogenous target DNA. The
extended region may also comprise other functional sequence elements, such as,
but not limited
to, a "primer binding site" and a "spacer or linker" sequence, or other
structural elements, such
as, but not limited to aptamers, stem loops, hairpins, toe loops (e.g., a 3'
toeloop), or an RNA-
protein recruitment domain (e.g., MS2 hairpin). As used herein the "primer
binding site"
comprises a sequence that hybridizes to a single-strand DNA sequence having a
3'end generated
from the nicked DNA of the R-loop.
1227] In certain embodiments, the PEgRNAs have a 5' extension arm, a spacer,
and a gRNA
core. The 5' extension further comprises in the 5' to 3' direction a reverse
transcriptase template,
a primer binding site, and a linker. The reverse transcriptase template may
also be referred to
more broadly as the "DNA synthesis template" where the polymerase of a prime
editor described
herein is not an RT, but another type of polymerase.
[228] In certain other embodiments, the PEgRNAs have a 5' extension arm, a
spacer, and a
gRNA core. The 5' extension further comprises in the 5' to 3' direction a
reverse transcriptase
template, a primer binding site, and a linker. The reverse transcriptase
template may also be
referred to more broadly as the "DNA synthesis template" where the polymerase
of a prime
editor described herein is not an RT, but another type of polymerase.
[229] In still other embodiments, the PEgRNAs have in the 5' to 3' direction a
spacer (1), a
gRNA core (2), and an extension arm (3). The extension arm (3) is at the 3'
end of the PEgRNA.
The extension arm (3) further comprises in the 5' to 3' direction a "homology
arm," an "edit
template," and a "primer binding site." In certain embodiments, a PEgRNA
comprises from 5' to
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3', a space, a DNA synthesis template, and a primer binding site. The
extension arm (3) may also
comprise an optional modifier region at the 3' and 5' ends, which may be the
same sequences or
different sequences. In addition, the 3' end of the PEgRNA may comprise a
transcriptional
terminator sequence. These sequence elements of the PEgRNAs are further
described and
defined herein.
[230] In still other embodiments, the PEgRNAs have in the 5' to 3' direction
an extension arm
(3), a spacer (1), and a gRNA core (2). The extension arm (3) is at the 5' end
of the PEgRNA.
The extension arm (3) further comprises in the 3' to 5' direction a "homology
arm," an "edit
template," and a "primer binding site." The extension arm (3) may also
comprise an optional
modifier region at the 3' and 5' ends, which may be the same sequences or
different sequences.
The PEgRNAs may also comprise a transcriptional terminator sequence at the 3'
end. These
sequence elements of the PEgRNAs are further described and defined herein.
PE1
[231] As used herein, "PEI" refers to a prime editor system comprising a
fusion protein
comprising Cas9(H840A) and a wild type MMLV RT having the following structure:
[NLS]-
[Cas9(H840A)]-[linker]-[MMLV RT(wt)] + a desired PEgRNA, wherein the PE fusion
has the
amino acid sequence of SEQ ID NO: 100, which is shown as follows;
MKRTADGSEP __ SPKKKRK'VDKKYSIGLDIGTNSVGWAVITDE YKVPSKICFICVLGNTDRHSIKKNLIGA
LLFDSGETAEATRLKRTARRRYTRRKNRIC'YLQUFSNEMAKVDDSFFIIRLEESFLVEEDICKHERHPI
FGNI'VDEVAYHEICYPTIYILLRKKL'VDSTDKADLRLIYLALAHMIKFRGHELIEGDLNPDNSDVDKLFI
QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN
FDIAEDAKLQLSKRTYDDDLDNLLAQIGDQYADLFLAAKNISDAILLSDILRVNTEITKAPLSASMIKR
YDEIIIIQDLTLLKALVRQQLPEKYKEIFFDQSICNGYAGYIDGGASQEEFYKIIKPILEKMDGTEELLV
KLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYP.FLKDNREICIEICILTFRIPYYVGPLARGNS
RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKRSLLYEYFTVYNELTKV
KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRICVTVKQLKE D YFICKIECFDSVEISGVEDRFNASLGTY
HDLLKIIKDKDFLDNEENEDIL.EDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLICRRRYTGWGR
LSRICLINGIRDKQSGICTIL.DFLKSDGFANRNFMQLIEIDDSLTFICEDIQ.KAQVSGQGDSLHERIANLAG
SPAIKKGILQTVICVVDELVICVMGRRKPENIVIEMARENQTTQKGQICNSRERMICRIEEGIKELGSQ1L
KEHPVENTQLQNEKLYL Y YLQN GRDMYVDQELDINRLSD YDVDAIVPQSFLKDDSIDNKVLTRSDKN
RGKSDNVPSEEVVKKIVIKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGEIKRQLVETR(NTK
HVAQILDSRMNT1CYDENDICLIREVKVITLKSKLVSDFRXDFQFYKVRE1NNYHHAIIDAYLNAVVGTA
LIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN1MNFFKTETTLANGEIRICRPLIET
NGETGEIVWDKGRDFATVRKVLSMPQVN1VICKTEVQTGGTSKESILPKRNSDKUARKKOWDPKKY
GGFDSPTVAYSVLVVAKVEKGKS1CKLKSVICELLGITIMERSSFEKNP1DFLEAKGYKEVICKDLIII:LP
KYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQIIKII Y
LDELIEQISEFSKRVILADANLDICVLSAYN KIIRDKPIREQAEN MILFILTNLGAPAAFKYFDTTIDRICR
YTSTKEVLDATLILIQSITGL 'YETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSILME
DEYRLIIE'ISKEPDVS'LGSTWLSDI;PQAWAETGGMGLAVRQAPLHPLKATSTPVSIKQYPMSQEARLGIKPIHQRL
L
DQGILVPCQSPWNTPLLPVKKPG'ThDYRPVQDLREVNKRVEDIHPTVPNPYNLLS'GLPPSHQWYTfrIDLKDAFFC

LRLHPTSQPLFAFEWRDPEI4GB`GQLTIVTRLPQGFICAISPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATS

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ELDCQQGTRA LLQTLGNLGYRASAKKAQICQKQVICYLGYLLKEGQRWLTEARKETIMGQPTPKTPRQLREFLGT
AGFCRLWTPGFA.EMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELF'VDEKQGYAKGV
LTQICLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGICLTMGQPLVILAPHAVEALVKQPPDRWIõSN
A RMTHYQALLLDTDRVOFGPVVA LNPA TLLPLPEEGLQHNCLDILA
EAHGTRPDLTDQPLPDADH7'WYT'DGSSL
LQEGQRKAGAAPTTETEVIWAKALPAGTSAQRAELIALTQALKAMEGICKLNVY7'DSRYA
FATAHIHGEIYRRRGLL
TSEGKEIKNKDEJLALLKALFLPKRLSHHCPGHQKGHSAEARGNRMADQAARKAAITET.'PDTSTLLIENSSPSGGS

ICRTADGSEFE.PKKKIIKV (SEQ 113 NO: 100)
KEY:
NUCLEAR. LOCALIZATION SEOUENCE (NLS) TOP:(SEQ ID NO: 101), BOTTOM: (SEQ ID NO:
103)
CAS9(11840A) (SEQ ID NO: 37) (SEQ ID NO: 37 corresponds identically to SEQ ID
NO: 2, except with an H840A
substitution)
33-AMINO ACID LINKER (SEQ. ID NO: 102)
M-IVILV reverse transcriptase (SEQ ID NO: 81).
[232] Alternatively, PE1 may also refer to the prime editor fusion protein of
SEQ m NO: 100,
i.e., without the pegRNA complexed thereto. PEI may be complexed with a pegRNA
during
operation and/or use in prime editing.
PE2
[233] As used herein, "PE2" refers to a prime editing system comprising a
fusion protein
comprising Cas9(H840A) and a variant MNILV RT having the following structure:
[NLS]-
[Cas9(H840AW[1inker]-[MMLV_RT(D200N)(7330P)(L603W)(T306K)(W313F)] + a desired
PEgRNA, wherein the PE fusion has the amino acid sequence of SEQ ID NO: 107,
which is
shown as follows:
MKRTADGSEFESPKICKRKVDICKYSIGLDIGTNSVGWAVITDEYKVPSICKFICVLGNTDRHSIKKNLIGA
LLFDSGEMEATRLICRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERIIPI
FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAIIMIKFRGHFLIEGDLNPDNSDVDKLFI
QLVQTYNQLFEENPINASGVDAKMISARLSKSRRLENLIAQLPGEKICNGLFGNLIALSLGLTPNFKSN
FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNISDAILLSDILRVNTEITKAPLSASM1KR
YDEMIQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEICMDGTEELLV
ICLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLICDNREKIEKILTFRIPYYVGPIARGNS
RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEICVLPKILSLLYEYFTVYNELTKV
KYVTEGMRKPAFISGEQICKAIVDLLFKTNRKVTVKQLKEDYFICKIECFDSVEISGVEDRFNASLGTY
HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR
ISRICLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLIIEFIIANLAG
SPAIKKGILQTVICVVDELVKVMGRHKPENIVIEMARENQTTQKGQICNSRERMKRIEEGIKELGSQIL
KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFIKDDSIDNICVLTRSDKN
RGKSDNVPSEEVVICICMKNYWRQLLNAICLITQRKFDNLTICAERGGLSELDICAGFIKRQLVETRQITK
EIVAQILDSRMNTKYDENDKLIREVKVITLICSICINSDFRICDFQFYKVREINNYEIHAHDAYLNAVVGTA
LIKKYPICLESEFVYGDYKVYDVRKMIAKSEQEIGKATAICYFFYSNIMNFFKTEITLANGEIRKRPLIET
NGETGEIVWDKGRDFATVRKVISMPQVNIVICKTEVQTGGFSKESILPKRNSDKLIARKICDWDPKKY
GGFDSPTVAYSVLVVAICVEKG.KSKKLKSVKELLGMMERSSFEKNPIDFLEAKGYKEVKKDLIIKLP
KYSLFELENGRKRMIASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKFIY
LDEHEQISEFSKR.VILADANLDKVISAYNKFIRDICPIREQAENIIRLFTLTNLGAPAAFKYFDITIDRKR
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YTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLN/E
DEYRLHETSKEP DLSIESTWLSDFPQA WA ETGGMGLAVRQAPLI IP LKA TSTPVSIKQY PMSQ EA
RLGIKPHIQRLL
DQGILVPCQSPW ATTP LLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG L PPSHQ WYTYLDLKDA
FFC
LRLHPTSQ PLFAFEWRDPEMGISGQLTYPTRLPQGFKNSPTLFNEALHRDLA DFRIQHPDLILLQYVDDLLLAA
TSE
LDCQQGTRALLQTLENLGYRASAKKAQICQKQVKYLGYLLKEGORWLTEA RKETYMGQPTPKTPRQLREFLGKA
GFCRLFIPGFA.EMAAPLYPL.TKPGTLFNWGPDQQKAYQEIKQALLTAPA LGLPDLTKPFELFVDEKQGYAKGVLT

QKLGP WRRPVAY LSKKLDPVAAGWPPCIRMVA AIAVLTKDAG KLTMGQ P LVI LA PHAVEALVKQ
PPDRWLSNAR
MTHYQA LLIDTDRVQFGPVVA LNPATLLPL PEEGLQ HNCI.DILA EA HGTRPDLIDQPLPDA
DIITWYTDGSSLLQ
EGQRKAGAA VITETEVIWA KALPAGTSA ORA EUALTOALKMA EGKKL NVIITDSRYA FA TA H IHGEIY
RRRGWL TS'
EGKEIKNKDE LALL KALFLPKRISIILICPGIIQKGHSAEA RGNRMADQAARKAAITETPDTSTL
LIEMSSPSGGSKR
TADGSEFEPKKKRKV (SEQ ID NO: 107)
KEY:
NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 101), BOTTOM: (SEQ ID NO:
103)
CAS9(11840A) (SEQ ID NO: 37)
33-AMINO ACID LINKER (SEO ID NO: 102)
M-M1.1" reverse transcriptase (SEQ ID NO: 98).
12341 Alternatively, PE2 may also refer to the prime editor fusion protein of
SEQ ID NO: 107,
i.e., without the pegRNA complexed thereto. PE2 may be complexed with a pegRNA
during
operation and/or use in prime editing.
PE3
[235] A.s used herein, "PE3" refers to PE2 plus a second-strand nicking guide
RNA that
complexes with the PE2 and introduces a nick in the non-edited DNA strand in
order to induce
preferential replacement of the edited strand.
PE3b
[236] As used herein, "PE3b" refers to PE3 but wherein the second-strand
nicking guide RNA
is designed for temporal control such that the second strand nick is not
introduced until after the
installation of the desired edit. This is achieved by designing a gRNA with a
spacer sequence
that- matches only the edited strand, but not the original allele. Using this
strategy, referred to
hereafter as PE3b, mismatches between the protospacer and the unedited allele
should disfavor
nicking by the sgRNA. until after the editing event on the PAM strand takes
place.
PE4
12371 As used herein, "PE4" refers to a system comprising PE2 plus an MLH1
dominant
negative protein (ix., wild-type ML:Hl. with amino acids 754-756 truncated as
described further
herein) expressed in trans. In some embodiments, PE4 refers to a fusion
protein comprising PE2
and an MLII1 dominant negative protein joined via an optional linker.
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PE5
[238] As used herein, "PE5" refers to a system comprising PE3 plus an MLH1
dominant
negative protein (i.e., wild-type MLH1 with amino acids 754-756 deleted as
described further
herein, which may be referred to as "M_LH1 A754-756" or "MLHIdn") expressed in
trans. In
some embodiments, PE5 refers to a fusion protein comprising PE3 and an MLH1
dominant
negative protein joined via an optional linker.
PEmax
[239] As used herein, "PEmax" (see FIG. 54B) refers to a PE complex comprising
a fusion
protein comprising Cas9(R221K N394K H840A) and a variant MMLV RT pentamutant
(13200N
T306K W313F T330P L603W) having the following structure: [bipartite NLS]-
[Cas9(12221K)(N394K)(H840A)Mlinker]-[MMLV_RT(D200N)(T330P)(L603W)Hbipartite
NLSHNLS] + a desired PEgRNA, wherein the PE fusion has the amino acid sequence
of SEQ
ID NO: 99.
PE4max
[240] As used herein, "PE4max" refers to PE4 but wherein the PE2 component is
substituted
with PEmax.
PE5max
[241] As used herein, "PE5max" refers to PE5 but wherein the PE2 component of
PE3 is
substituted with PEmax.
PE-short
[2421 As used herein, "PE-short" refers to a PE construct that is fused to a C-
terminally
truncated reverse transcriptase, and has the following amino acid sequence:
NIKRTADGSEFESPKKKRKVDKKYSIGLDIG'TNSVGWAVITDEYKVPSKKFKVLGNTDRIISIKKNLIGA
LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEEFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI
FGNIVDEVAYHEKYPT1YRLRKKLVDSTDICADLRLIYLALARMIKFRGIIFLIEGDLNPDNSDVDICLFI
QLVQTYNQLFEENPINASGVDAICAIL,SARLSKSRRLENLIAQLPGEICKNGLFGNL1ALSLGLTPNFKSN
FDIAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAICNISDAILISD1LRVNTEITKAPLSASMIKR
YDEIIRQDLTLLKAL'VRQQLPEICYKEIFIFDQS1CNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLV
ICLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF'YPFLKDNREKIEKILTFRIPYITVGPLARGNS
RFAWMTRKSEET1TPWNFEEV'VDKGASAQSFIERMTNFDKNLPNEKVLPICHSLLYEYFTVYNELTKV
KYVTEGMRKPAFLSGEQKKANDLLFKTNRKVTVKQLICEDYFICICIECFDS'VEISGVEDRFNASLGTY
IIDLLICIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDICVMKQLKRRRYTGWGR
LSRKLINGIRDKQSGICTILDFLKSDGFANRNFMQLIHDDSLTFICEDIQICAQVSGQGDSLHEHIANLAG
SPAIKKGILQTVICVVDELVKVMGRHICPENIVIEMARENQTTQKG'QICNSRERMKRIEEGIKELGSQ1L
ICERPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD'YDVDAIVPQSFLICDDSIDNKVLTRSDKN
RGKSDN'VPSEEVVIOCMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG1FIKRQLVETRQITIC
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HVAQILDSRMNTKVDENDKLIREVICVITI,KSKINSDFRKDFQVYKVRETNNYHHAHDAYLNAVVGTA
LIKKATKLESEFVYGDYKVYDVRKNIIAKSEQEIGKATAKYFFYSNEVINFFKTEITLANGEIRKRPLIET
NGETGEIVWDKGRDFA.TVRKVLSMPQVNIVKKTEVQTGGFSKESELPKRNSDIKILIARKKDWDPKKY
GGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDIATKI2
KYSLFELENGRKIR1VHASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQI.FVEQHMIY
LDEITEQISEFSKRVILADANIDKVISAYNKFIRDICPIREQAENITHLFTITNLGAPAAFKYFDTTIDRKR
YTST.KEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLN/E
DEYR1JTh7.'SKEPDVSLGS7W1SDFPQAWAETGGMGLAVRQAPLHPIXATSTPVSIKQYP.MSQEARLGIKPHIQRL
L
DQGILVPCQSPWN7'PLLPVKKPGTND.YRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFC
LRLHPTSQPLFAFEWRDPEMCHSGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIOHPDLILLOYVDDLLLAATSE
LDCQQGTRALLQTLGNLGYRASAKKAQK'QKQVKYLGYLIXEGQRWLTF,ARKETBYGQPTPKTPROLREFLGKA
GFCRLFIPGFAEMAAPLYPLTKPGTI,F'NWGPDQQKAYOEIKQALL7:4PALGLPDLTKPFELFVDEKQGYAKGVLT

QKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVIIAPHAVEALVKQPPDRWLSNAR
MTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDNSRLINSGGSKRTADGSEFEPKKKRK.V (SEQ
ID NO: 117)
KEY:
NUCLEAR LOCALIZATION SEQUENCE (NLS) TOP:(SEQ ID NO: 101), BOTTOM: (SEQ ID NO:
103)
CAS9(H840A) (SEQ ID NO: 37)
33-AM1NO ACID LINKER 1 (SEQ ID NO: 102)
M-MLV TRUNCATED reverse tremscriptase (SEQ ID NO: 80)
Polymerase
[243] As used herein, the term "polymerase" refers to an enzyme that
synthesizes a nucleotide
strand and that may be used in connection with the prime editor systems
described herein. The
polymerase can be a "template-dependent" polymerase (i.e., a polymerase that
synthesizes a
nucleotide strand based on the order of nucleotide bases of a template
strand). The polymerase
can also be a "template-independent" polymerase (i.e., a polymerase that
synthesizes a
nucleotide strand without the requirement of a template strand). A polymerase
may also be
further categorized as a "DNA polymerase" or an "RNA polymerase." In various
embodiments,
the prime editor system comprises a DNA polymerase. In various embodiments,
the DNA
polymerase can be a "DNA-dependent DNA polymerase" (i.e., whereby the template
molecule is
a strand of DNA). In such cases, the DNA template molecule can be a PEgRNA,
wherein the
extension arm comprises a strand of DNA. In such cases, the PEgRNA may be
referred to as a
chimeric or hybrid PEgRNA which comprises an RNA portion (i.e., the guide RNA
components,
including the spacer and the gRNA core) and a DNA portion (i.e., the extension
arm). In various
other embodiments, the DNA polymerase can be an "RNA-dependent DNA polymerase"
(i.e.,
whereby the template molecule is a strand of RNA). In such cases, the PEgRNA
is RNA, i.e.,
including an RN.A extension. The term "polymerase" may also refer to an enzyme
that catalyzes
the polymerization of nucleotide (i.e., the polymerase activity). Generally,
the enzyme will
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initiate synthesis at the 3'-end of a primer annealed to a polynucleotide
template sequence (e.g.,
such as a primer sequence annealed to the primer binding site of a PEgRNA) and
will proceed
toward the 5' end of the template strand. A "DNA polymerase" catalyzes the
polymerization of
deoxynucleotides. As used herein in reference to a DNA polymerase, the term
DNA polymerase
includes a "functional fragment thereof." A "functional fragment thereof'
refers to any portion of
a wild-type or mutant DNA polymerase that encompasses less than the entire
amino acid
sequence of the polymerase and which retains the ability, under at least one
set of conditions, to
catalyze the polymerization of a polynucleotide. Such a functional fragment
may exist as a
separate entity, or it may be a constituent of a larger polypeptide, such as a
fusion protein.
Prime editing
[244] As used herein, the term "prime editing" refers to an approach for gene
editing using
napDNAbps, a polymerase (e.g., a reverse transcriptase), and specialized guide
RNAs that
include a DNA synthesis template for encoding desired new genetic information
(or deleting
genetic information) that is then incorporated into a target DNA sequence.
Certain embodiments
of prime editing are described in the embodiments of FIG. 1. Classical prime
editing is described
in the inventors' publication of Anzalone, A. V. et al. Search-and-replace
genome editing
without double-strand breaks or donor DNA. Nature 576, 149-157(2019), which is
incorporated
herein by reference in its entirety.
12451 Prime editing represents a platform for genome editing that is a
versatile and precise
genome editing method that directly writes new genetic information into a
specified DNA site
using a nucleic acid programmable DNA binding protein ("napDNAbp") working in
association
with a polymerase (i.e., in the form of a fusion protein or otherwise provided
in trans with the
napDNAbp), wherein the prime editing system is programmed with a prime editing
(PE) guide
RNA ("PEgRNA") that both specifies the target site and templates the synthesis
of the desired
edit in the form of a replacement DNA strand by way of an extension (either
DNA or RNA)
engineered onto a guide RNA (e.g., at the 5' or 3' end, or at an internal
portion of a guide RNA).
The replacement strand containing the desired edit (e.g., a single nucleobase
substitution) shares
the same (or is homologous to) sequence as the endogenous strand (immediately
downstream of
the nick site) of the target site to be edited (with the exception that it
includes the desired edit).
Through DNA repair and/or replication machinery, the endogenous strand
downstream of the
nick site is replaced by the newly synthesized replacement strand containing
the desired edit. In
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some cases, prime editing may be thought of as a "search-and-replace" genome
editing
technology since the prime editors, as described herein, not only search and
locate the desired
target site to be edited, but at the same time, encode a replacement strand
containing a desired
edit which is installed in place of the corresponding target site endogenous
DNA strand. The
prime editors of the present disclosure relate, in part, to the mechanism of
target-primed reverse
transcription (TPRT), which can be engineered for conducting precision
CRISPR/Cas-based
genome editing with high efficiency and genetic flexibility. TPRT is used by
mobile DNA
elements, such as mammalian non-LIR retrotransposons and bacterial Group II
introns. The
inventors have herein used Cas protein-reverse transcriptase fusions or
related systems to target a
specific DNA sequence with a guide RNA, generate a single strand nick at the
target site, and
use the nicked DNA as a primer for reverse transcription of an engineered
reverse transcriptase
template that is integrated with the guide RNA. However, while the concept
begins with prime
editors that use reverse transcriptase as the DNA polymerase component, the
prime editors
described herein are not limited to reverse transcriptases but may include the
use of virtually any
DNA polymerase. Indeed, while the application throughout may refer to prime
editors with
"reverse transcriptases," it is set forth here that reverse transcriptases are
only one type of DNA
polymerase that may work with prime editing. Thus, wherever the specification
mentions a
"reverse transcriptase," the person having ordinary skill in the art should
appreciate that any
suitable DNA polyinerase may be used in place of the reverse transcriptase.
Thus, in one aspect,
the prime editors may comprise Cas9 (or an equivalent napDNAbp) which is
programmed to
target a DNA sequence by associating it with a specialized guide RNA (i.e.,
PEgRNA)
containing a spacer sequence that anneals to a complementary protospacer in
the target DNA.
The specialized guide RNA also contains new genetic information in the form of
an extension
that encodes a replacement strand of DNA containing a desired genetic
alteration which is used
to replace a corresponding endogenous DNA strand at the target site. To
transfer information
from the PEgRNA to the target DNA, the mechanism of prime editing involves
nicking the target
site in one strand of the DNA to expose a 3'-hydroxyl group. The exposed 3'-
hydroxyl group can
then be used to prime the DNA polymerization of the edit-encoding extension on
PEgRNA
directly into the target site. In various embodiments, the extension¨which
provides the template
for polymerization of the replacement strand containing the edit¨can be formed
from RNA or
DNA. In the case of an RNA extension, the polymerase of the prime editor can
be an RNA-
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dependent DNA. polymerase (such as, a reverse transcriptase). In the case of a
DNA extension,
the polymerase of the prime editor may be a DNA-dependent DNA polymerase. The
newly
synthesized strand (i.e., the replacement DNA strand containing the desired
edit) that is formed
by the herein disclosed prime editors would be homologous to the genomic
target sequence (i.e.,
have the same sequence as) except for the inclusion of a desired nucleotide
change (e.g., a single
nucleotide change, a deletion, or an insertion, or a combination thereof). The
newly synthesized
(or replacement) strand of DNA may also be referred to as a single strand DNA
flap, which
would compete for hybridization with the complementary homologous endogenous
DNA strand,
thereby displacing the corresponding endogenous strand. In certain
embodiments, the system can
be combined with the use of an error-prone reverse transcriptase enzyme (e.g.,
provided as a
fusion protein with the Cas9 domain, or provided in trans to the Cas9 domain).
The error-prone
reverse transcriptase enzyme can introduce alterations during synthesis of the
single strand DNA
flap. Thus, in certain embodiments, error-prone reverse transcriptase can be
utilized to introduce
nucleotide changes to the target DNA. Depending on the error-prone reverse
transcriptase that is
used with the system, the changes can be random or non-random. Resolution of
the hybridized
intermediate (comprising the single strand DNA flap synthesized by the reverse
transcriptase
hybridized to the endogenous DNA strand) can include removal of the resulting
displaced flap of
endogenous DNA (e.g., with a 5' end DNA flap endonuclease, FEN1), ligation of
the synthesized
single strand DNA flap to the target DNA, and assimilation of the desired
nucleotide change as a
result of cellular DNA repair and/or replication processes. Because templated
DNA synthesis
offers single nucleotide precision for the modification of any nucleotide,
including insertions and
deletions, the scope of this approach is very broad and could foreseeably be
used for myriad
applications in basic science and therapeutics.
[246] In various embodiments, prime editing operates by contacting a target
DNA molecule
(for which a change in the nucleotide sequence is desired to be introduced)
with a nucleic acid
programmable DNA binding protein (napDNAbp) complexed with a prime editing
guide RNA
(PEgRNA). In various embodiments, the prime editing guide RNA (PEgRNA)
comprises an
extension at the 3' or 5' end of the guide RNA, or at an intramolecular
location in the guide RNA
and encodes the desired nucleotide change (e.g., single nucleotide change,
insertion, or deletion).
In step (a), the napDNAbp/extended gRNA complex contacts the DNA molecule, and
the
extended gRNA guides the napDNAbp to bind to a target locus. In step (b), a
nick in one of the
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strands of DNA of the target locus is introduced (e.g., by a nuclease or
chemical agent), thereby
creating an available 3' end in one of the strands of the target locus. In
certain embodiments, the
nick is created in the strand of DNA that corresponds to the R-loop strand,
i.e., the strand that is
not hybridized to the guide RNA sequence, i.e., the "non-target strand." The
nick, however,
could be introduced in either of the strands. That is, the nick could be
introduced into the R-loop
"target strand" (i.e., the strand hybridized to the protospacer of the
extended gRNA) or the "non-
target strand" (i.e., the strand forming the single-stranded portion of the R-
loop and which is
complementary to the target strand). In step (c), the 3' end of the DNA strand
(formed by the
nick) interacts with the extended portion of the guide RNA in order to prime
reverse
transcription (i.e., "target-primed RT"). In certain embodiments, the 3' end
DNA strand
hybridizes to a specific RT priming sequence on the extended portion of the
guide RNA, i.e., the
"reverse transcriptase priming sequence" or "primer binding site" on the
PEgRNA. In step (d), a
reverse transcriptase (or other suitable DNA polymerase) is introduced which
synthesizes a
single strand of DNA from the 3' end of the primed site towards the 5' end of
the prime editing
guide RNA. The DNA polymerase (e.g., reverse transcriptase) can be fused to
the napDNAbp or
alternatively can be provided in trans to the napDNAbp. This forms a single-
strand DNA flap
comprising the desired nucleotide change (e.g., the single base change,
insertion, or deletion, or a
combination thereof) and which is otherwise homologous to the endogenous DNA
at or adjacent
to the nick site. In step (e), the napDNAbp and guide RNA are released. Steps
(f) and (g) relate
to the resolution of the single strand DNA flap such that the desired
nucleotide change becomes
incorporated into the target locus. This process can be driven towards the
desired product
formation by removing the corresponding 5' endogenous DNA flap that forms once
the 3' single
strand DNA flap invades and hybridizes to the endogenous DNA sequence. Without
being bound
by theory, the cells endogenous DNA repair and replication processes resolves
the mismatched
DNA to incorporate the nucleotide change(s) to form the desired altered
product. The process
can also be driven towards product formation with "second strand nicking."
This process may
introduce at least one or more of the following genetic changes:
transversions, transitions,
deletions, and insertions.
[247] The term "prime editor (PE) system" or "prime editor (PE)" or "PE
system" or "PE
editing system" refers the compositions involved in the method of genome
editing using target-
primed reverse transcription (TPRT) describe herein, including, but not
limited to the
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napDNAbps, reverse transcriptases (or another DNA polymerase), fusion proteins
(e.g.,
comprising napDNAbps and reverse transcriptases or comprising napDNAbps and
DNA
polymerases), prime editing guide RNAs, and complexes comprising fusion
proteins and prime
editing guide RNAs, as well as accessory elements, such as second strand
nicking components
(e.g., second strand sgRNAs) and 5' endogenous DNA flap removal endonucleases
(e.g., FEN I)
for helping to drive the prime editing process towards the edited product
formation.
[2481 Although in the embodiments described thus far the PEgRNA constitutes a
single
molecule comprising a guide RNA (which itself comprises a spacer sequence and
a gRNA core
or scaffold) and a 5' or 3' extension arm comprising the primer binding site
and a DNA synthesis
template, the PEgRNA may also take the form of two individual molecules
comprised of a guide
RNA and a trans prime editor RNA template (tPERT), which essentially houses
the extension
arm (including, in particular, the primer binding site and the DNA synthesis
domain) and an
RNA-protein recruitment domain (e.g., MS2 aptamer or hairpin) in the same
molecule which
becomes co-localized or recruited to a modified prime editor complex that
comprises a tPERT
recruiting protein (e.g., MS2cp protein, which binds to the MS2 aptamer).
Prime editor
[249] The term "prime editor" refers to fusion constructs comprising a
napDNAbp (e.g., Cas9
nickase) and a reverse transcriptase and is capable of carrying out prime
editing on a target
nucleotide sequence in the presence of a PEgRNA (or "extended guide RNA"). The
term "prime
editor" may refer to the fusion protein or to the fusion protein complexed
with a PEgRNA,
and/or further complexed with a second-strand nicking sgRNA. In some
embodiments, the prime
editor may also refer to the complex comprising a fusion protein (reverse
transcriptase fused to a
napDNAbp), a PEgRNA, and a regular guide RNA capable of directing the second-
site nicking
step of the non-edited strand as described herein. In certain embodiments, a
prime editor (e.g.,
PE1, PE2, or PE3) may be provided as a system along with an inhibitor of the
DNA mismatch
repair pathway, such as a dominant negative MLH I protein. In various
embodiments, the
inhibitor of the DNA mismatch repair pathway, such as a dominant negative MLH1
protein, may
be provided in trans to the prime editor. In other embodiments, the inhibitor
of the DNA
mismatch repair pathway, such as a dominant negative MLH1 protein, may be
complexed to the
prime editor, e.g., coupled through a linker to the prime editor fusion
protein.
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Primer binding site
1250] The term "primer binding site" or "the PBS" refers to the portion of a
PEgRNA as a
component of the extension arm (for example, at the 3' end of the extension
arm) . The term
"primer binding site" refers to a single-stranded portion of the PEgRNA as a
component of the
extension arm that comprises a region of complementarity to a sequence on the
non-target strand.
In some embodiments, the primer binding site is complementary to a region
upstream of a nick
site in a non-target strand. In some embodiments, the primer binding site is
complementary to a
region immediately upstream of a nick site in the non-target strand. In some
embodiments, the
primer binding site is capable of binding to the primer sequence that is
formed after nicking (e.g.,
by a nickase component of a prime editor, for example, a Cas9 nikcase) of the
target sequence by
the prime editor. When a prime editor nicks one strand of the target DNA
sequence (e.g., by a
Cas nickase component of the prime editor), a 3'-ended ssDNA flap is formed,
which serves a
primer sequence that anneals to the primer binding site on the PEgRNA to prime
reverse
transcription. In some embodiments, the PBS is complementary to, or
substantially
complementary to, and can anneal to, a free 3' end on the non-target strand of
the double
stranded target DNA at the nick site. In some embodiments, the PBS annealed to
the free 3' end
on the non-target strand can initiate target-primed DNA synthesis.
Protospacer
12511 As used herein, the term "protospacer" refers to the sequence (-20 bp)
in DNA adjacent
to the PAM (protospacer adjacent motif) sequence. The protospacer shares the
same sequence as
the spacer sequence of the guide RNA. The guide RNA anneals to the complement
of the
protospacer sequence on the target DNA (specifically, one strand thereof,
i.e., the "target strand"
versus the "non-target strand" of the target DNA sequence). In some
embodiments, in order for a
Cas nickase component of the prime editor to function it also requires a
specific protospacer
adjacent motif (PAM) which varies depending on the Cas protein component
itself, e.g., the type
of Cas protein. For example, the most commonly used Cas9 nuclease, derived
from S. pyogenes,
recognizes a PAM sequence of NGG that is found directly downstream of the
target sequence in
the genomic DNA, on the non-target strand. The skilled person will appreciate
that the literature
in the state of the art sometimes refers to the "protospacer" as the ¨20-nt
target-specific guide
sequence on the guide RNA itself, rather than referring to it as a "spacer."
Thus, in some cases,
the term "protospacer" as used herein may be used interchangeably with the
term "spacer." The
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context of the description surrounding the appearance of either "protospacer"
or "spacer" will
help inform the reader as to whether the term is in reference to the ,gRNA or
the DNA target.
Protospacer adjacent motif (PAM)
12521 As used herein, the term "protospacer adjacent sequence" or "PAM" refers
to an
approximately 2-6 base pair DNA sequence that is an important targeting
component of a Cas9
nuclease. Typically, the PAM sequence is on either strand, and is downstream
in the 5' to 3'
direction of the Cas9 cut site. The canonical PAM sequence (i.e., the PAM
sequence that is
associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9) is 5'-
NGG-3' wherein
"N" is any nucleobase followed by two guanine ("G") nucleobases. Different PAM
sequences
can be associated with different Cas9 nucleases or equivalent proteins from
different organisms.
In addition, any given Cas9 nuclease, e.g., SpCas9, may be modified to alter
the PAM specificity
of the nuclease such that the nuclease recognizes alternative PAM sequence.
1.2531 For example, with reference to the canonical SpCas9 amino acid sequence
is SEQ ED
NO: 2, the PAM sequence can be modified by introducing one or more mutations,
including (a)
D1135V, R1335Q, and T1337R "the VQR variant", which alters the PAM specificity
to NGAN
or NGNG, (b) D1135E, R1335Q, and T1337R "the EQR variant", which alters the
PAM
specificity to NGAG, and (c) D1135V, G1218R, R1335E, and T1337R "the VRER
variant",
which alters the PAM specificity to NGCG. In addition, the D1 135E variant of
canonical SpCas9
still recognizes NGG, but it is more selective compared to the wild type
SpCas9 protein.
12541 lIt will also be appreciated that Cas9 enzymes from different bacterial
species (i.e., Cas9
orthologs) can have varying PAM specificities. For example, Cas9 from
Staphylococcus aureus
(SaCas9) recognizes NGRRT or NGRRN. In addition, Cas9 from Neisseria
meningitis (NmCas)
recognizes NNNNGATT. In another example, Cas9 from Streptococcus thermophilis
(StCas9)
recognizes NNAGAAW. In still another example, Cas9 from Treponema denticola (T
dCas)
recognizes NAAAAC. These are examples and are not meant to be limiting. It
will be further
appreciated that non-SpCas9s bind a variety of PAM sequences, which makes them
useful when
no suitable SpCas9 PAM sequence is present at the desired target cut site.
Furthermore, non-
SpCas9s may have other characteristics that make them more useful than SpCas9.
For example,
Cas9 from Staphylococcus aureus (SaCas9) is about 1 ldlobase smaller than
SpCas9, so it can be
packaged into adeno-associated virus (AAV). Further reference may be made to
Shah et al.,
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"Protospacer recognition motifs: mixed identities and functional diversity,"
RNA Biology, 10(5):
891-899 (which is incorporated herein by reference).
Reverse transcriptase
[255] The term "reverse transcriptase" describes a class of polymerases
characterized as RNA-
dependent DNA polymerases. All known reverse transcriptases require a primer
to synthesize a
DNA transcript from an RNA template. Historically, reverse transcriptase has
been used
primarily to transcribe mRNA into cDNA which can then be cloned into a vector
for further
manipulation. Avian myoblastosis virus (AMV) reverse transcriptase was the
first widely used
RNA-dependent DNA polymerase (Verma, Biochim. Biophys. Acta 473:1 (1977)). The
enzyme
has 5'-3' RNA.-directed DNA polymerase activity, 5'-3' DNA-directed DNA
polymerase activity,
and RNase IH activity. RNase H is a processive 5' and 3' ribonuclease specific
for the RNA
strand for RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning,
New York:
Wiley & Sons (1984)). Errors in transcription cannot be corrected by reverse
transcriptase
because known viral reverse transcriptases lack the 3'-5' exonuclease activity
necessary for
proofreading (Saunders and Saunders, Microbial Genetics Applied to
Biotechnology, London:
Croom Helm (1987)). A detailed study of the activity of A.MV reverse
transcriptase and its
associated RNase H activity has been presented by Berger etal., Biochemistry
22:2365-2372
(1983). Another reverse transcriptase which is used extensively in molecular
biology is reverse
transcriptase originating from Moloney murine leukemia virus (M-MLV). See,
e.g., Gerard, G.
R., DNA 5:271-279 (1986) and Kotewicz, M. L., etal., Gene 35:249-258 (1985). M-
MLV
reverse transcriptase substantially lacking in RNase H activity has also been
described. See, e.g.,
U.S. Pat. No. 5,244,797. The invention contemplates the use of any such
reverse transcriptases,
or variants or mutants thereof.
[256] In addition, the invention contemplates the use of reverse
transcriptases that are error-
prone, i.e., that may be referred to as error-prone reverse transcriptases or
reverse transcriptases
that do not support high fidelity incorporation of nucleotides during
polymerization. During
synthesis of the single-strand DNA flap based on the RT template integrated
with the guide
RNA, the error-prone reverse transcriptase can introduce one or more
nucleotides which are
mismatched with the RT template sequence, thereby introducing changes to the
nucleotide
sequence through erroneous polymerization of the single-strand DNA flap. These
errors
introduced during synthesis of the single strand DNA flap then become
integrated into the double
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strand molecule through hybridization to the corresponding endogenous target
strand, removal of
the endogenous displaced strand, ligation, and then through one more round of
endogenous DNA
repair and/or sequencing processes.
Reverse transcription
12571 As used herein, the term "reverse transcription" indicates the
capability of an enzyme to
synthesize a DNA strand (that is, complementary DNA or cDNA) using RNA as a
template. In
some embodiments, the reverse transcription can be "error-prone reverse
transcription," which
refers to the properties of certain reverse transcriptase enzymes which are
error-prone in their
DNA polymerization activity.
Protein, peptide, and polypeptide
[2581 The terms "protein," "peptide," and "polypeptide" are used
interchangeably herein, and
refer to a polymer of amino acid residues linked together by peptide (amide)
bonds. The terms
refer to a protein, peptide, or polypeptide of any size, structure, or
function. Typically, a protein,
peptide, or polypeptide will be at least three amino acids long. A protein,
peptide, or polypeptide
may refer to an individual protein or a collection of proteins. One or more of
the amino acids in a
protein, peptide, or polypeptide may be modified, for example, by the addition
of a chemical
entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a
farnesyl group, an
isofarnesyl group, a fatty acid group, a linker for conjugation,
finctionalization, or other
modification, etc. A protein, peptide, or polypeptide may also be a single
molecule or may be a
multi-molecular complex. A protein, peptide, or polypeptide may be just a
fragment of a
naturally occurring protein or peptide. A protein, peptide, or polypeptide may
be naturally
occurring, recombinant, or synthetic, or any combination thereof. Any of the
proteins provided
herein may be produced by any method known in the art. For example, the
proteins provided
herein may be produced via recombinant protein expression and purification,
which is especially
suited for fusion proteins comprising a peptide linker. Methods for
recombinant protein
expression and purification are well known, and include those described by
Green and
Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor
Laboratory
Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are
incorporated herein by
reference.
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Silent mutation
[259] As used herein, the term "silent mutation" refers to a mutation in a
nucleic acid molecule
that does not have an effect on the phenotype of the nucleic acid molecule, or
the protein it
produces if it encodes a protein. Silent mutations can be present in coding
regions of a nucleic
acid (i.e., segments of a gene that encode for a protein), or they can be
present in non-coding
regions of a nucleic acid. A silent mutation in a nucleic acid sequence, e.g.,
in a target DNA
sequence or in a DNA synthesis template sequence to be installed in the target
sequence, may be
a nucleotide alteration that does not result in expression or function of the
amino acid sequence
encoded by the nucleic acid sequence, or other functional features of the
target nucleic acid
sequence. When silent mutations are present in a coding region, they may be
synonymous
mutations. Synonymous mutations refer to substitutions of one base for another
in a gene such
that the corresponding amino acid residue of the protein produced by the gene
is not modified.
This is due to the redundancy of the genetic code, allowing for multiple
different codons to
encode for the same amino acid in a particular organism. When a silent
mutation is in a non-
coding region or a junction of a coding region and a non-coding region (e.g.,
an intron/exon
junction), it may be in a region that does not impact any biological
properties of the nucleic acid
molecule (e.g., splicing, gene regulation, RNA lifetime, etc.). Silent
mutations may be useful, for
example, for increasing the length of an edit made to a target nucleotide
sequence using prime
editing to evade correction of the edit by the MA4R pathway as described
herein. In certain
embodiments, the number of silent mutations installed may be one, or two, or
three, or four, or
five, or six, or seven, or eight, or nine, or ten, or more. In certain other
embodiments involving
at least two silent mutations, the silent mutations may be installed within
one, or two, or three, or
four, or five, or six, or seven, or eight, or nine, or ten, or 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21,
22, 23, 24, or 25 nucleotides from the intended edit site.
Spacer sequence
[2601 As used herein, the term "spacer sequence" in connection with a guide
RNA or a
PEgRNA refers to the portion of the guide RNA or PEgRNA of about 20
nucleotides (e.g., 16,
17, 18, 19, 20, 21, 22, 23 or 24 nucleotides) which contains a nucleotide
sequence that shares the
same sequence as the protospacer sequence in the target DNA sequence. The
spacer sequence
anneals to the complement of the protospacer sequence to form a ssRNAJssDNA
hybrid structure
at the target site and a corresponding R loop ssDNA structure of the
endogenous DNA strand.
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Target site
1261] The term "target site" refers to a sequence within a nucleic acid
molecule to be edited by
a prime editor (PE) disclosed herein. The target site may refer to the
endogenous sequence within
the nucleic acid molecule to be edited, e.g., endogenous genomic sequence of a
target genome,
which is identical to the sequence of the DNA synthesis template except for
the one or more
nucleotide edits to be installed present on the DNA synthesis template (and
except that the DNA
synthesis template contains Uracil instead of Thymine), or the corresponding
endogenous
sequence on the non-target strand that is complementary to the DNA synthesis
template except
for one or more mismatches at the position of the one or more nucleotide edits
to be installed
present on the DNA synthesis template. The target site may also further refer
to the sequence
within a nucleic acid molecule to which a complex of the prime editor (PE) and
gRNA binds.
Variant
[262] As used herein the term "variant" should be taken to mean the exhibition
of qualities that
have a pattern that deviates from what occurs in nature, e.g., a variant Cas9
is a Cas9 comprising
one or more changes in amino acid residues as compared to a wild type Cas9
amino acid
sequence. The term "variant" encompasses homologous proteins having at least
75%, or at least
80%, or at least 85%, or at least 90%, or at least 95%, or at least 99%
percent identity with a
reference sequence and having the same or substantially the same functional
activity or activities
as the reference sequence. The term also encompasses mutants, truncations, or
domains of a
reference sequence, and which display the same or substantially the same
functional activity or
activities as the reference sequence.
Vector
[263] The term "vector," as used herein, refers to a nucleic acid that can be
modified to encode
a gene of interest and that is able to enter into a host cell, mutate and
replicate within the host
cell, and then transfer a replicated form of the vector into another host
cell. Exemplary suitable
vectors include viral vectors, such as retroviral vectors or bacteriophages
and filamentous phage,
and conjugative plasmids. Additional suitable vectors will be apparent to
those of skill in the art
based on the instant disclosure.
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DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
12641 The present disclosure provides compositions and methods for prime
editing with
improved editing efficiency and/or reduced indel formation by inhibiting the
DNA mismatch
repair pathway while conducting prime editing of a target site. The inventors
have surprisingly
found that the editing efficiency of prime editing may be significantly
increased (e.g., 2-fold
increase, 3-fold increase, 4-fold increase, 5-fold increase, 6-fold increase,
7-fold increase, 8-fold
increase, 9-fold increase, 10-fold increase, 11-fold increase, 12-fold
increase,13-fold increase,
14-fold increase, 15-fold increase, 16-fold increase, 17-fold increase, 18-
fold increase, 19-fold
increase, 20-fold increase, 21-fold increase, 22-fold increase, 23-fold
increase, 24-fold increase,
26-fold increase, 27-fold increase,28-fold increase, 29-fold increase, 30-fold
increase, 31-fold
increase, 32-fold increase, 33-fold increase, 34-fold increase, 35-fold
increase, 36-fold increase,
37-fold increase, 38-fold increase, 39-fold increase, 40-fold increase ,41-
fold increase, 42-fold
increase, 43-fold increase, 44-fold increase, 45-fold increase, 46-fold
increase, 47-fold increase,
48-fold increase, 49-fold increase, 50-fold increase, 51-fold increase, 52-
fold increase, 53-fold
increase, 54-fold increase, 55-fold increase, 56-fold increase, 57-fold
increase, 58-fold increase,
59-fold increase, 60-fold increase, 61-fold increase, 62-fold increase, 63-
fold increase, 64-fold
increase, 65-fold increase, 66-fold increase, 67-fold increase, 68-fold
increase, 69-fold increase,
70-fold increase, 71-fold increase, 72-fold increase, 73-fold increase, 74-
fold increase, 75-fold
increase, 76-fold increase, 77-fold increase, 78-fold increase, 79-fold
increase, 80-fold increase,
81-fold increase, 82-fold increase, 83-fold increase, 84-fold increase, 85-
fold increase, 86-fold
increase, 87-fold increase, 88-fold increase, 89-fold increase, 90-fold
increase, 91-fold increase,
92-fold increase, 93-fold increase, 94-fold increase, 95-fold increase, 96-
fold increase, 97-fold
increase, 98-fold increase, 99-fold increase, 100-fold increase or more) when
one or more
functions of the DNA mismatch repair (MMR) system are inhibited, blocked, or
otherwise
inactivated during prime editing. In addition, the inventors have surprisingly
found that the
frequency of indel formation resulting from prime editing may be significantly
decreased (e.g.,
2-fold decrease, 3-fold decrease, 4-fold decrease, 5-fold decrease, 6-fold
decrease, 7-fold
decrease, 8-fold decrease, 9-fold decrease, or 10-fold decrease or lower) when
one or more
functions of the IDN.A mismatch repair (MMR) system are inhibited, blocked, or
otherwise
inactivated during prime editing.
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[2651 The disclosure relates to the surprising finding that the efficiency
and/or specificity of
prime editing is impacted by a cell's own DNA mismatch repair (MAIR) DNA
repair pathway.
As described herein (e.g., in Example 1), the inventors developed a novel
genetic screening
method---referred to in one embodiment as "pooled CRISPRi screen for prime
editing
outcomes"¨which led to the identification of various genetic determinates,
including MMR, as
affecting the efficiency and/or specificity of prime editing. Accordingly, in
one aspect, the
present disclosure provides novel prime editing systems comprising a means for
inhibiting and/or
evade the effects of MMR, thereby increasing the efficiency and/or specificity
of prime editing.
In one embodiment, the disclosure provides a prime editing system that
comprises an MMR-
inhibiting protein, such as, but not limited to, a dominant negative MMR
protein, such as a
dominant negative MLH1 protein (i.e., "MLHldn"). In another embodiment, the
prime editing
system comprises the installation of one or more silent mutations nearby an
intended edit,
thereby allowing the intended edit from evading MMR recognition, even in the
absence of an
MMR-inhibiting protein, such as an MLHldn. In another aspect, the disclosure
provides a novel
genetic screen for identifying genetic determinants, such as MMR, that impact
the efficiency
and/or specificity of prime editing. In still further aspects, the disclosure
provides nucleic acid
constructs encoding the improved prime editing systems described herein. The
disclosure in
other aspects also provides vectors (e.g., AAV or lentivirus vectors)
comprising nucleic acids
encoding the improved prime editing system described herein. In still other
aspects, the
disclosure provides cells comprising the improved prime editing systems
described herein. The
disclosure also provides in other aspects the components of the genetic
screens, including nucleic
acid and/or vector constructs, guide RNA, pegRNAs, cells (e.g., CRISPRi
cells), and other
reagents and/or materials for conducting the herein disclosed genetic screens.
In still other
aspects, the disclosure provides compositions and kits, e.g., pharmaceutical
compositions,
comprising the improved prime editing system described herein and which are
capable of being
administered to a cell, tissue, or organism by any suitable means, such as by
gene therapy,
mRNA delivery, virus-like particle delivery, or ribonucleoprotein (RNP)
delivery. In yet another
aspect, the present disclosure provides methods of using the improved prime
editing system to
install one or more edits in a target nucleic acid molecule, e.g., a genomic
locus. In still another
aspect, the present disclosure provides methods of treating a disease or
disorder using the
improved prime editing system to correct or otherwise repair one or more
genetic changes (e.g., a
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single polymorphism) in a target nucleic acid molecule, e.g., a genomic locus
comprising one or
more disease-causing mutations.
[266] In one embodiment, the MLI-11 protein is inhibited, blocked, or
otherwise inactivated. In
other embodiments, other proteins of the MMR system are inhibited, blocked, or
otherwise
inactivated, including, but not limited to, PMS2 (or MutL alpha), PMS1 (or
MutL beta), MLH3
(or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6,
PCNA, RFC, EX01, POLS, and PCNA. The inhibition may involve inhibiting the
protein with
an inhibitor (e.g., antibody or small molecule inhibitor or a dominant
negative variant of the
protein which disrupts, blocks, or otherwise inactivates the function of the
protein, e.g., a
dominant negative form of MLII1). The inhibition may also involve any other
suitable means,
such as by protein degradation (e.g., PROTAC-based degradation of MLH1),
transcript-level
inhibition (e.g., siRNA transcript degradation / gene silencing or microRNA-
based inhibition of
translation of the MLH1 transcript), or at the genetic level (i.e., installing
a mutation in the
MLHI gene (or regulatory regions) which inactivates or reduces the expression
of the MLHI
gene, or which installs a mutation which inactivates, blocks, or minimizes
that activity of the
encoded MLH1 product). In addition, the disclosure contemplates that the prime
editor (e.g.,
delivered as a fusion protein comprising a napDNAbp and a polymerase, such as
a Cas9 nickase
fused to a reverse transcriptase) may be administered together with any
inhibitor of the DNA
mismatch repair pathway.
[267] Accordingly, the present disclosure provides a method for editing a
nucleic acid molecule
by prime editing that involves contacting a nucleic acid molecule with a prime
editor, a pegRNA,
and an inhibitor of the DNA mismatch repair pathway, thereby installing one or
more
modifications to the nucleic acid molecule at a target site with increased
editing efficiency and/or
lower indel formation. The present disclosure further provides polynucleotides
for editing a
DNA target site by prime editing comprising a nucleic acid sequence encoding a
napDNAbp, a
polymerase, and an inhibitor of the DNA mismatch repair pathway, wherein the
napDNAbp and
polymerase is capable in the presence of a pegRNA of installing one or more
modifications in
the DNA target site with increased editing efficiency and/or lower indel
formation. The
disclosure further provides, vectors, cells, and kits comprising the
compositions and
polynucleotides of the disclosure, as well as methods of making such vectors,
cells, and kits, as
well as methods for delivery such compositions, polynucleotides, vectors,
cells and kits to cells
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in vitro, ex vivo (e.g., during cell-based therapy which modify cells outside
of the body), and in
vivo.
MMR pathway
12681 As noted above, the present disclosure relates to the observation that
the efficiency and/or
specificity of prime editing is impacted by a cell's own DNA mismatch repair
(MMR) DNA
repair pathway. DNA mismatch repair (MMR) is a highly conserved biological
pathway that
plays a key role in maintaining genomic stability (e.g., see FIG. 8A and 8B).
Escherichia colt
MutS and Mutl, and their eukaryotic homologs, MutSa and MutLa, respectively,
are key players
in MMR-associated genome maintenance. In various aspects, the disclosure
contemplates any
suitable means by which to inhibit, block, or otherwise inactivate the DNA
mismatch repair
(MMR) system, including, but not limited to inactivating one or more critical
proteins of the
MMR. system at the genetic level, e.g., by introducing one or more mutations
in the gene(s)
encoding a protein of the MMR system. Such proteins include, but are not
limited to MLH1,
PMS2 (or MutL alpha), PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS alpha
(MSH2-
MSH6), MutS beta (MSH2-MSI-13), MSH2, MSH6, PCNA, RFC, EX01, POLO, and PCNA.
The
nucleotide and amino acid sequences of such naturally occurring proteins and
variants thereof
are known in the art. Exemplary sequences are provided herein. The present
disclosure embraces
using any inhibitor, blocking agent, knockdown strategy, or other means of
inactivating any
known protein involved in MMR ("MMR protein"), including any wild type or
naturally
occurring variant of such MMR protein, and any engineered variant (including
single or multiple
amino acid substitutions, deletions, insertions, rearrangements, or fusions)
of such MMR protein,
so long as the inhibiting, blocking, or otherwise inactivation of one or more
of said M:MR
proteins or variants thereof result in the inhibition, blockage, or
inactivation of the MMR
pathway. The inhibiting, blocking, or inactivation of any one or more MMR
proteins or variants
may be by any suitable means applied at the genetic level (e.g., in the gene
encoding the one or
more MMR proteins, such as introducing a mutation that inactivates the MMR
protein or variant
thereof), transcriptional level (e.g., by transcript knockdown), translational
level (e.g., by
blocking translation of one or more MMR proteins from their cognate
transcripts), or at the
protein level (e.g., administering of an inhibitor (e.g., small molecule,
antibody, dominant
negative protein variant) or by targeted protein degradation (e.g., PROTAC-
based degradation).
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[269] In one aspect, the present disclosure provides an improved method of
prime editing
comprising additionally inhibiting the DNA mismatch repair ('MMR) system
during prime
editing by inhibiting, blocking, or otherwise inactivating MLH1 or a variant
thereof.
[270] Without being bound by theory, MLH1 is a key MMR protein that
heterodirrierizes with
PMS2 to form MutL alpha, a component of the post-replicative DNA mismatch
repair system
(MMR). DNA repair is initiated by MutS alpha (MSH2-MSH6) or MutS beta (MSH2-
MSH3)
binding to a dsDNA mismatch, then MutL alpha is recruited to the heteroduplex.
Assembly of
the MutL-MutS-heteroduplex ternary complex in presence of RFC and PCNA is
sufficient to
activate endonuclease activity of PMS2. It introduces single-strand breaks
near the mismatch and
thus generates new entry points for the exonuclease EX01 to degrade the strand
containing the
mismatch. DNA methylation would prevent cleavage and therefore assure that
only the newly
mutated DNA strand is going to be corrected. MutL alpha (MLH1-PMS2) interacts
physically
with the clamp loader subunits of DNA polymerase III, suggesting that it may
play a role to
recruit the DNA polymerase Ill to the site of the MMR. Also implicated in DNA
damage
signaling, a process which induces cell cycle arrest and can lead to apoptosis
in case of major
DNA damages. MLH1 also heterodimerizes with MLH3 to form MutL gamma which
plays a
role in meiosis.
12711 The "canonical" human MLH1 amino acid sequence is represented by SEQ ID
NO: 204.
1272] MLH1 also may include other human isoforms, including P40692-2 (SEQ ID
NO: 205),
which differs from the canonical sequence in that residues 1-241 of the
canonical sequence are
missing.
[273] MLH1 also may include a third known isoform known as P40692-3 (SEQ ID
NO: 207),
which differs from the canonical sequence in that residues 1-101 (of
MSFVAG'VIRR...ASISTYGFRG (SEQ ID NO: 206)) are replaced with MAF.
MMR inhibitors and methods of IVIIVIR inhibition
[274] The present disclosure provides a method for editing a nucleic acid
molecule by prime
editing that involves contacting a nucleic acid molecule with a prime editor,
a pegRNA, and an
inhibitor of the DNA mismatch repair pathway, thereby installing one or more
modifications to
the nucleic acid molecule at a target site with increased editing efficiency
and/or lower indel
formation. Thus, the present disclosure contemplates any suitable means to
inhibit MMR. In one
embodiment, the disclosure embraces administering an effective amount of an
inhibitor of the
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MMR pathway. In various embodiments, the MMR pathway may be inhibited by
inhibiting,
blocking, or inactivating any one or more MMR proteins or variants at the
genetic level (e.g., in
the gene encoding the one or more MMR proteins, such as introducing a mutation
that
inactivates the MMR protein or variant thereof), transcriptional level (e.g.,
by transcript
knockdown), translational level (e.g., by blocking translation of one or more
MMR proteins from
their cognate transcripts), or at the protein level (e.g., application of an
inhibitor (e.g., small
molecule, antibody, dominant negative protein partner) or by targeted protein
degradation (e.g.,
PROTAC-based degradation). The present disclosure also contemplates methods of
prime
editing which are designed to install modifications to a nucleic acid molecule
that evade
correction by the MMR pathway, without the need to provide an MMR inhibitor.
12751 The inventors developed prime editing which enables the insertion,
deletion, or
replacement of genomic DNA sequences without requiring error-prone double-
strand DNA
breaks. The present disclosure now provides an improved method of prime
editing involving the
blocking, inhibiting, or inactivation of the MMR pathway (e.g., by inhibiting,
blocking, or
inactivating an MMR pathway protein, including MLH1) during prime editing,
whereby doing so
surprisingly results in increased editing efficiency and reduced indel
formation. As used herein,
"during" prime editing can embrace any suitable sequence of events, such that
the prime editing
step can be applied before, at the same time, or after the step of blocking,
inhibiting, or
inactivating the MMR pathway (e.g., by targeting the inhibition of MLH1).
12761 Prime editing uses an engineered Cas9 nickase¨reverse transcriptase
fusion protein (e.g.,
PE1 or PE2) paired with an engineered prime editing guide RNA (pegRNA) that
both directs
Cas9 to the target genomic site and encodes the information for installing the
desired edit. Prime
editing proceeds through a multi-step editing process: 1) the Cas9 domain
binds and nicks the
target genomic DNA site, which is specified by the pegRNA's spacer sequence;
2) the reverse
transcriptase domain uses the nicked genomic DNA as a primer to initiate the
synthesis of an
edited DNA strand using an engineered extension on the pegRNA as a template
for reverse
transcription¨this generates a single-stranded 3' flap containing the edited
DNA sequence; 3)
cellular DNA repair resolves the 3' flap intermediate by the displacement of a
5' flap species that
occurs via invasion by the edited 3' flap, excision of the 5' flap containing
the original DNA
sequence, and ligation of the new 3' flap to incorporate the edited DNA
strand, forming a
heteroduplex of one edited and one unedited strand; and 4) cellular DNA repair
replaces the
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unedited strand within the heteroduplex using the edited strand as a template
for repair,
completing the editing process.
[277] Efficient incorporation of the desired edit requires that the newly
synthesized 3' flap
contains a portion of sequence that is homologous to the genomic DNA site.
This homology
enables the edited 3' flap to compete with the endogenous DNA strand (the
corresponding 5'
flap) for incorporation into the DNA duplex. Because the edited 3' flap will
contain less
sequence homology than the endogenous 5' flap, the competition is expected to
favor the 5' flap
strand. Thus, a potential limiting factor in the efficiency of prime editing
may be the failure of
the 3' flap, which contains the edit, to effectively invade and displace the
5' flap strand.
Moreover, successful 3' flap invasion and removal of the 5' flap only
incorporates the edit on
one strand of the double-stranded DNA genome. Permanent installation of the
edit requires
cellular DNA repair to replace the unedited complementary DNA strand using the
edited strand
as a template. While the cell can be made to favor replacement of the unedited
strand over the
edited strand (step 4 above) by the introduction of a nick in the unedited
strand adjacent to the
edit using a secondary sgRNA (the PE3 system), this process still relies on a
second stage of
DNA repair.
[278] This disclosure describes a modified approach to prime editing that
comprises
additionally inhibiting, blocking, or otherwise inactivating the DNA mismatch
repair (MMR)
system. In some embodiments, an MMR inhibitor is provided to the target
nucleic acid along
with other components of a prime editing system, for example, an exogenous MMR
inhibitor
such as an siRNA can be provided to a cell comprising the target nucleic acid.
In some
embodiments, a prime editing system component, e.g., a pegRNA, is designed to
install
modifications in the target nucleic acid which evade the MMR system, without
the need to
provide an inhibitor. In certain embodiments, the DNA mismatch repair (MMR)
system can be
inhibited, blocked, or otherwise inactivating one or more proteins of the MMR
system,
including, but not limited to MLH1, PMS2 (or Muth alpha), PMSI (or MutL beta),
MLH3 (or
MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA,
RFC, EXO I, PODS, and PCNA. The disclosure contemplates any suitable means by
which to
inhibit, block, or otherwise inactivate the DNA mismatch repair (MMR) system,
including, but
not limited to inactivating one or more critical proteins of the MMR system at
the genetic level,
e.g., by introducing one or more mutations in the genes encoding a protein of
the MMR system,
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e.g., MLH1, PMS2 (or MutL alpha), PMSI (or MutL beta), MLII3 (or MutL gamma),
MutS
alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX0I, POL8,
and PCNA.
[279] Thus, in one aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating the DNA mismatch repair (MMR) system.
[2801 In another aspect, the present disclosure provides a method for editing
a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating a protein of the MMR system, e.g., MLH1, PMS2 (or MutL alpha),
PMS1 (or MutL
beta), MI.J13 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3),
MSH2, MSH6, PCNA, RFC, EX01, POLO, and PCNA.
[281] In one aspect, the present disclosure provides an improved method of
prime editing
comprising additionally inhibiting the DNA mismatch repair (MMR) system during
prime
editing by inhibiting, blocking, or otherwise inactivating MLH1 or a variant
thereof. Without
being bound by theory, MLH1 is a key MMR protein that heterodimerizes with
PMS2 to form
MutL alpha, a component of the post-replicative DNA mismatch repair system
(MMR). DNA
repair is initiated by MutS alpha (MSH2-MSH6) or MutS beta (MSFI2-MSH3)
binding to a
dsDNA mismatch, then MutL alpha is recruited to the heteroduplex. Assembly of
the MutL-
MutS-heteroduplex ternary complex in presence of RFC and PCNA is sufficient to
activate
endonuclease activity of PMS2. It introduces single-strand breaks near the
mismatch and thus
generates new entry points for the exonuclease EX01 to degrade the strand
containing the
mismatch. DNA methylation would prevent cleavage and therefore assure that
only the newly
mutated DNA strand is going to be corrected. MutL alpha (MLH1-PMS2) interacts
physically
with the clamp loader subunits of DNA polymerase III, suggesting that it may
play a role to
recruit the DNA polymerase III to the site of the MMR. Also implicated in DNA
damage
signaling, a process which induces cell cycle arrest and can lead to apoptosis
in case of major
DNA damages. MLH1 also heterodimerizes with MLH3 to form MutL gamma which
plays a
role in meiosis. The "canonical" human MLH1 amino acid sequence is represented
by SEQ ID
NO: 204
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[282] MLH1 also may include other human isoforms, including P40692-2 (SEQ ID
NO: 205),
which differs from the canonical sequence in that residues 1-241 of the
canonical sequence are
missing.
[283] MLH1 also may include a third known isoform known as P40692-3 (SEQ ID
NO: 207),
which differs from the canonical sequence in that residues 1-101 (of
MSFVAGVIRR...ASISTYGFRG (SEQ ID NO: 206)) are replaced with MAF.
[284] The disclosure contemplates that any of the following MLH1 proteins may
be inhibited
by an inhibitor, or otherwise blocked or inactivated in order to inhibit the
MMR pathway during
prime editing. In addition, such exemplary proteins may also be used to
engineer or otherwise
make a dominant negative variant that may be used as a type of inhibitor when
administered in
an effective amount which blocks, inactivates, or inhibits the MMR. Without
being bound by
theory, it is believed that MLH1 dominant negative mutants can saturate
binding of MutS.
Exemplary MLH1 proteins include the following amino acid sequences, or amino
acid sequences
having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or up to 100% sequence identity
with any of the
following sequences:
Description Sequence SEQ
ID NO:
MLH1 MSFVA.GVIRRLDETWNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGG 204
Homo sapiens LKLIQIQDNGTORKEDLDIVCERFTTSKLQSFEDLASISTYGFR.GEALASTSIIVAH
SwissProt VTITTKTADGKCAYRASYSDGKLICAPPKPCAGNQGTQITVEDLFYNIATRRICAL
Accession No. KNPSEEYGKILEVVGRYSVHNAGISFSVKK.QGETVADVRTLPNASTVDNIRSIFG
P40692 NAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFIA,FINHRLVE.STSLRKAM
Wild type TVYAAYL.PKNTHPFLYLSLEISPQNVDVNVHPTICHEVHFLHEESILERVQQIITES
ICLLGSNSSRMYFTQTLLPGLAGPSGEMVKSITSLTSSSTSGSSDKVYAIIQMVR.T
DSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVA
AKNQSLEGDTTK.GTSEMSEKR.GPTSSNPRKRHREDSDVEMVEDDSRICEMTAAC
TPRRRITNLTS'VLSLQEEINE,QGHEVLREMLHNHSFVGCVNPQWALAQHQTKLY
LLNITKLSEELFYQUAYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEEDGP
ICEGLAEYIVEF.LKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRL
ATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSE'VPGSIPNS'WKWT
VENIVYKALRSHILPPKHF thOGNILQLANLPDLYKVFERC
MLH1 MAFVAGVIRRLDETVVNRIAAGEVIQRPANAIICEMIENCLDAKSINIQVVVICEG 219
Mus muscu/us GLKLIQIQDNGTGIRICEDLDIVCERFTTSKLQWEDLASISTYGFRGEALASISIIV
SwissProt AFIVTITIKTADGKCAYRASYSDGICLQAPPICPCAGNQGTLITVEDLFYNIITRRK
Accession No. ALKNPSEEYGKILEVVGRYSLFINSGISFSVKKQGETVSDVRTLPNATTVDNIRSIF
Q9JK91 GNAVSRELIEVGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVE.SAALRK
Wild type AIETVYAAYLPICNTHPFLYLSLEISPQNVDVNVHPTICHEVHFLHEESILQRVQQH
IESKLLGSNSSRMYFTQTLLPGLAGPSGEAARPTTGVASSSTSGSGDKVYAYQM
VRTDSRF,QICL.DAFLQPVSSLGPSQPQDPAPVRGARTEGSPERATREDEEMLALP
APAEAAAESENLMESLMETSDAAQKAAPTSSPGSSRKRHREDSDVEMVEN A S
GKEMTAA.CYPRRRIINLTSVLSLQEEISERCHETLREMLRNHSFVGCVNPQWAL
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AQHQTKLYLLNTTICLSEELF'YQILIYDFANFGVLRLSEPAPLFDLAMLALDSPES
G'WTEDDGPKEGLAEYIVEFLKKICAEMLADYFSVEIDEEGNLIGLPLLIDSYVPPL
EGLPIFILRLATEVNVVDEEKECFESLSKECA1VIFYSIRKQYILEESTLSGQQSDMPG
STSICPWKWIVEHITYKAF'RSHILPPKHFTE'DGNVLQLANLPDLYKVFERC
=
MLHi
MSFVAGVIRRIDETVVNRIAAGEVIQRPANAIICEMTENCIDAKSTNIQVIVREG 220
Rattus GLKLIQIQDNGTGIRKEDLD1VCERFTTSKLQ EFEDLAMISTYGFRGEALASISHV
norvegicus AHVTITTKTADGKCAYRASYSDGICLQAPPKPCAGNQGTLITVEDLFYNIrFRKK
SwissProt ALKNPSEEYGKILEVVGRYSIHNSGISFSVKICQGETVSDVRTLPNATTVDNJRSIF
Accession No. GNAVSRELIEVGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESAALKK
P97679 ATEAVYAAYLPKNTHPFLYLTLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHI
Wild type ESKLLGSNSSRMYFTQTLLPGLAGPSGEAVKSTTGIASSSTSGSGDKVHAYQMV
WIDSRDQKLDAFMQPVSRRLPSQPQDPVPGNRTEGSPEKAMQKDOEISELPAPM
EAAADSASLERESVIGASEVVAPQRHPSSPGSSRKRHPEDSDVEMMENDSRICEM
TAACYPRRRIINLTSVLSLQEEINDRGHETLREMLRNHTFVGC'VNPQWALAQHQ
TKLYLLNITKLSEELFYQILIYDFANFGVLRLPEPAPLFDFAMLALDSPESGWTE
EDGPKEGLAEYIVEFLKKKAKMLAD'YFS'VEIDEEGNLIGLPLLIDSYVPPLEGLPI
FILRLATEVNWDEEECTESL SKECAVFYS1RKQYILEESALSGQQSDMPGSPSKP
WKWTVEHIIYKAFRSHLLPPKHFTEDGNVLQLANLPDLCKVFERC
MLH1 MSLVAGVIRIILDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVVVKEG 221
Bos taurus GLICLIQIQDNGTOIRKEDLEIVCERFTTSKLQSFEDLAHISTYGFRGEALASISHV
SwissProt AHVTITIXTADGKCAYRAHYSDGKLKAPPICPCAGNQGTQUVEDLFYNISTRRIC
Accession No. ALKNPSFEYGKILEVVGRYAVHNSGIGFSVKKQGETVADVRTI,PNATTVDNIRS
F IMPGO IFGNAVSRELIEVECEDK.TLAFKMNGYISNANYSVKKCIFILFINHRINESASLRK
Wild type ATETVYAAYLPKSTHPFIXLSLEISPQNVDVNVHPTKHEVHFLHEDSILERLQQHI
ESRLLGSNASRTYFTQTLLPGLPGPSGEAVKSTASVTSSSTAGSGDRVYAHQMV
R.TDCREQKLDAFLQPVSKALSSQPQAVVPEHRTDA.SSSGTRQQDEEMLELPAPA
AVAAKSQALEDDATMRAADLAEICRGPSSSPENPRKRPRF,DSDVEMVEDASRKE
MTA.ACTPRRRIENLTSVISLQEEINERGBETLREMLENHSFVGCVNPQWALA.QH
QTKLYLLN'TTRLSEELFYQIIõVYDFANFGVLRLSF..PAFLFDLAMLALDSPESGWT
EEDGPKEGLAEYIVEFLKICKAEMLADYFSLEIDEEGNLVGLPLL1DNYVPPLEGL
PIFILRLATEVNWDFEKECFESISKECAMFYSIRKQYVSAE:STLSGQQSEVPGST
ANPWKWTVEHVIYKAFRSHLLPPKHFTEDGNILQLANLPDLYKVFFIZC
[2851 The methods and compositions described herein utilize MLH1 mutants or
truncated
variants. In some embodiments, the mutants and truncated variants of th.e
human MLIII wild-
type protein are utilized.
[286] in one aspect, a truncated variant of human. MLH1 is provided by this
disclosure. In some
embodiments, amino acids 754-756 of the wild-type human MLH1 protein are
truncated (6.754-
756, hereinafter referred to as MLHldn). In some embodiments, a truncated
variant of human
MLHI comprising only the N-terminal domain (amino acids 1-335) is provided
(hereinafter
referred to as MLH1 dem. In various embodiments, the following MLH1 variants
are provided
in this disclosure:
Description Sequence SEQ
ID NO:
MLH1 E34A MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKAMIENCLDAKSTSIQVIVICEGG 222
LKLIQIQDNGTGIRKEDLDIVCERFTISICLQSFEDLASISTYGFRGEALASISHVAH
VTITTKTADGKCAYRASYSDGKLICAPPKPCAGNQGTQIT'VEDLFYNIATRRICAL
KNPSEEYGKELEVVGRYSVIINAGISFSVICKQGFIVADVRTLPNASTVDNIRSIFG
NAVSRELIEIGCEDKTLAFKMNGYISNANYSVKICCIFLLFINHRLVESTSLRKAIE
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TVYAAYLPKNTHPFLYL SLEISPQNVD VNVHPTICEIEVHFLHEESILER VQQHIES
ICLLGSNS SRIvIYFTQTLLPGL AGP SGEM VKSTI' SLT S S ST SGS SDK VYAHQMVRT
DSREQICLDAFLQPL SKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVA
AKNQSLEGDITICGTSEMSEKRGPTSSNPRKRHREDSDVEMVEDDSRICEMTAAC
TPRRRIINLTS'VLSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQTKLY
LLNITKLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWiEEDGP
ICEGLAE'YIVEFLICKICAEMLADYFSLEIDEMNLIGLPLUDNYVPPLEGLP1FILRL
ATEVNWDEEICECFESLSICECAMFYSTRKQYISEESTLSGQQSEVPGSIPNSWKWT
VEHIVYKALRSHILPPICHF IEDGNILQLANLPDLYKVFERC
.MLH I E756 MSEVAGVIRRLDETVVNRIAAGEVIQRPANATICEIViTENCLDAKSTSIQVIVKEGG 208
LKLIQIQDNGTGIRKEDLDIVCERFTTSICLQSFEDLASISTYGFRGEALASISHVAH
VTITTKTADGKCAYRASYSDGKLKAP.PKPCAGNQGTQITVEDLFYNIATRRKAL
KNPSEEYGICTLE'VVGRYS'VHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFG
NAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRL'VESTSLRKAIE
TVYAAYLPICNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIES
KLLGSNSSRIvIYFTQTLLPGLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRT
DSREQICLDAFLQPLSKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVA
AKNQSLEGDTTICGTSENLSEKRGPTSSNPRKRHREDSD'VEMVEDDSRKEMTAAC
TPRRRTINLTSVLSLQEETN.EQGHEVLREMLHNHSFVCICVNPQWALAQHQTKLY
LLNTTKLSEELFYQILIYDFANFG'VLRLSEPAPLFDLAMLALDSPESGWTEEDGP
KEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPEFILRL
ATE VNWDEEKECFESLSKECAMFY SIRICQYISEESILSGQQSEVPGSIPNSWKWT
VEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVFERH
MLH I A754- MSFNAGVIRRLDETVVNRIAA.GEVIQRPANAIICEMIENCLDAKSTSIQVIVICEGG 209
756 LKLIQIQDNGTGIRKEDLDIVCERFTTSICLQSFEDLASISTYGFRGEALASISHVAH
VTITTKTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKAL
KNPSEEYGKTLEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSTFG
NAVSRELIEIGCEDKTLAFICMNGYISNANYSVKK CIFLLFINHRLVESTSLRICAIE
TVYAAYLPKNTHPFLYI,SLEISPQNVDVNVIIPTKHEVHFLHFISILERVQQHIES
KLLGSNSSRMYFTQTLLPGLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRT
DSREQKLDAFLQPI,SKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVA
AICNQSLEGD111(GTS:a4SEICRGPTSSNPRKRHREDSDVEMVEDDSRKEMTAAC
TPRRRTINLTSVLSLQEE1NEQGHEVLREIVILFINHSFVGCVNPQWALAQHQTKLY
LLNT.TKLSERT FYQILTYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEEDGP
KEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRL
ATEVNWDEEICECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWT
VEHIVYKALRSHTLPPICHFTEDGNILQLANLPDLYKVF1- - -1
MLH1 E34A MSEVAGVIRRLDETVVNRIAAGEVIQRPANATICAMIENCLDAKSTSIQVIVKEGG 210
A754-756 LKLIQIQDNGTORKEDLDIVCERFTTSKLQSFEDLASISTYGERGEALASISHVAH
VTITTKTADGKCAYRASYSDGKLKAPPKPCAGNQGTQTTVEDLFYNIATRRKAL
KNPSEEYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFG
NAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIE
TVYAAYLPKNTHPFLYLSLEISPQNVDVNVIIPTICHEVHFLITEESILERVQQHEES
KLLGSNSSRMYFTQTLLPGLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRT
DSREQKLDAFLQPLSKPLSSQPQAIVTEDK.TDISSGRARQQDEEMLELPAPAEVA
AKNQSLEGDTTKGTSEMSEKRGPTSSNPRKRHREDSDVEMVEDDSRICEMTA.AC
TPRRRIINLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQTKLY
LLNITKLSJEtLFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEEDGP
KEGLAEYIVEFLKICKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRI,
ATEVNWDEFJCECFESLSKECAMFYSIRKQYISEESTT.,SGQQSEVPGSIPNSWKWT
VEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVFT- - -1
MLHI 1-335 MSFVAGVIRI2LDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGG 211
LKLIQIQDNGTGIRKEDLDIVCERFITSKLQSFEDLASISTYGFRGEALASISHVAH
VITITKTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLEYNIATRRKAL
KNPSEEYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFG
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NAVSRELffiIGCEDKTLAFKMNGYISNANYS'VKKCIFLLFINHRLVESTSLRKAIE
TVYAAYLPKNTHPFLYL SLEISPQNVDVNVHPTKEIEVHFLHEESILER VQQHIES
KLL
MLH I. 1-335 MSFVAGVIRRLDETWNRIAAGEVIQRPANAIKA.MIENCLDAKSTSIQVIVKEGG 212
E34A LKLIQIQDNGTGIRKEDLDIVCERFITSICLQSFEDLA SISTYGFRGEALA.SISHVAH
VTITIXTADGKCAYRA SYSD GKLKAPPKPCAGNQGTQITVEDLFYNIATRRKAL
KNPSFXYGKILEVVGRYSVHNAGISFSVKKQGETVADVR.TLPNASTVDNIRSIFG
NA.VSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIE
TVY AAYLPKNTHPFLYL SLEISPQNVDVNVHPTKHEVHFLHEE SILERVQQHIES
KLL
MLH I 1-335 MSFVAGVIRRLDETVVNRIAAGEVIQRPANA1KEMIENCLDAK STSIQVI VICE GG 213
Nie ssvo LKLIQIQDNGTG1RKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAH
V=KTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIA'FRRKAL
ICNPSEEYGKILEVVGRY SVHNAGISFSVKKQGETVADVRTLPNASTVDNIRS1FG
NAVSRELIEIGCEDKTLAFKIANGYISNANYSVKKCIFLLFINIIRLVESTSLRKAIE
TVY AAYLPKNTHPFLYL SLEISPQNVDVNVHPTKFIEVHFLHEE SILERVQQHIE S
KLLPICKKRICV
WTI 1501-756 INLTS VL SLQEEINEQGHEVLREMLHNHSFVGC VNPQ WALAQHQTKLYLL NTT 215
KLSEELFYQ1L1YDFANFGVLRLSEPAPLFDLAMLALDSPESGW rhE,DGPKEGLA
EYIVEFLKKKAEIVILADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVN
WDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIV
YKALRSHILPPKHFTEDGNILQLANLPDLYKVFERC
MLH 1 501-753 INLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQTKLYLLN'rr 216
KLSFXLFYQIIIYDFANFGVLRLSEPAPLFDL AMLALDSPESGW11 ,DGPKEGLA
EYIVEFLKKKAEML ADYFSLE1DEEGNLIGLPLLIDNYVPPLEGLPIFILRL ATEVN
WDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGS1PNSWICWTVEIIIV
YKALRSHILPPKHFTEDGNILQL ANLPDLYKVF [- - -1
MLH1 461-756 KRGYISSNPRKRHREDSDVEIVIVEDDSRKEMTAACTPRRRIINLTSVLSLQEEINE 217
QGHEVLREMLHNHSFV GCVNPQWALAQHQTKLYLLNTTKL SEELFYQILIYDF
ANFG VLRLSEPAPLFDL AIVILALD SPE SGWTEEDGPKEGLAEYIVEFLKKKAEML
ADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESLSKE
CAIVIFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYKALRSHILPPIGIFT
MGNILQLANLPDLYKVFERC
MLH I 461-753 KRGPTSSNPRKR HREDSDVEMVPDD SRKEMTAACTPRRRUNLTSVLSLQEEINE 218
QGHEVLREMLIINHSFVGCVNPQWALAQHQ1KLYLLNITKLSEEL.FYQILIYDF
ANFGVIAL SEPAPLFDLAMLALD SPE SGWTEEDGPKEGLAEYIVFYIICKKAEML
ADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESLSKE
CAMFYSIRKQYISEESTLSGQQSEVPGSIPNSVVKWTVEHIVYKALRSHILPPKHFT
EDGNILQLANLPDLYK - -1
NLS8v" MLH1 P KKKRKV IN LTS VL SLQEE1NEQGHEVL REMLHNHSFVGCVNPQWALAQHQT 223
501-753 KL YLLNTTKLSEELFYQ1LIYDFANFGVLRLSEPAPLFDLA1VILALDSPESGWTEE
DGYKEGLAFYIVEFLICKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFI
1_,RL ATEVNVVDEEKECTESLSICECAMFYSIRKQYISEESTLSGQQSEVPGSWNSW
K WTVEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVFL- -
NL Ss V4 MLH1 PKKICRKVKRGPTSSNPRKRHREDSD'VEMVEDD SRKEMTAACTPRRRIINLTSV 224
461-753 L SLQEEINEQGHE'VLREMLHNHSPVGCVNPQWALAQHQTKLYLLNTTKL SEEL
FYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEF
LK KKAEML ADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEK
ECFESLSKECAMFY SIRKQYISEESTLS GQQSEVPGSIPNSWKWTVEHT VYK ALR
SHILPPICHFTEDGNILQLANLPDLYKVPF -
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[287] In another aspect, the present disclosure provides a method for editing
a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating PMS2 (or Mut', alpha) or variant thereof.
[288] In yet another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating PMS1 (or MutL beta) or variant thereof.
[289] In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating MLH3 (or MutL gamma) or variant thereof.
[290] In another aspect, the present disclosure provides a method for editing
a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating MutS alpha (MSH2-MSH6) or variant thereof.
[291] In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating MSH2 or variant thereof.
[2921 In another aspect, the present disclosure provides a method for editing
a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating MSH6 or variant thereof.
[293] In yet another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating PCNA or variant thereof.
[294] In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating RFC or variant thereof.
[295] In another aspect, the present disclosure provides a method for editing
a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating EXOlor variant thereof.
[296] In yet another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating POL8 or variant thereof.
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[297] Exemplary amino acid sequences for these MMR. proteins (PMS2 (or MutL
alpha),
PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta
(MSH2-MSH3), MS1712, MSH6, PCNA, RFC, EX01, POLE, and PCNA) are as follows:
Description Sequence
SEQ
ID
NO:
PMS2 MERAESSSTEPAKAIKPIDRKSVHQ1CSGQV'VLSLSTAVKELVENSLDAGATNIDL 225
Homo sapiens KLKDYGVDLIEVSDNGCGVEE.ENFEGLTLKHHTSKIQEFADLTQVETFGFRGEAL
SwissProt SSLCALSDVTISTCHASAKVGTRLMFDHNGKIIQKTPYPRPRGTTVSVQQLFSTLP
Accession No. VRHKEFQRNIKKEYAKMVQVLHAYCIISAGIR VSCTNQLGQGKRQPVVCTGGSP
P54278 &MEN IGS VFGQKQLQSLIPFVQLPPSDSVCEEY GLSCSDALHNLFYISGFISQCTHG
Wild type VGRSSTDRQFFFINRRPCDPAK VCRLVNE'VYHMYNRHQYPFVVLNISVDSECVDI
NVTPDKRQILLQEEKLLLAVLKTSLIGMFDSDVNICLNVSQQPLLDVEGNLIK.MH
AADLP.X.PMVEKQDQSPSLRTGELICKDVSISRLREAFSLRHTTENKPHSPKTPEPR
RSPLCiQKRGMLSSSTSGAISDKGVLRF'QKEAVSSSHGPSDPIDRAEVEICDSGHGS
TSVDSEGFSIPDTGSHCSSEY AASSPGDRGSQEHVDSQEKAPKTDDSFSD VDCHS
NQEDTGCKFRVLPQPINLATPNTICRFICKE'EILSSSDICQICL VNTQDMSASQVDVA
VKINKK'VVPLDFSMSSLAKRIKQLHHEAQQSEGEQNYRKFRAKICPGENQAAED
ELRKEISKTMFAEMEIIGQFNLGFITTKLNEDIFIVDQHATDEKYNFEMLQQHTVL
QGQRLIAPQTLNLTAVNEAVLIENLEIFRICNGFDIWIDENAPVTERAICLISLPTSICN
WTFGPQDVDELIFMLSDSPGVMCRPSRVKQMFASRACRKSVIVIIGTALNTSEMK
ICLITHMGEMDHPWNCPHGRPTMRHIANLGVISQN
PMS1 MKQLPAATVRLLSSSQIITSVVSVVKELIENSLDAGATSVDVKLENYGFDKIEVRD 226
Homo sapiens NGEGIKA VD APVMAMKYYTSKINSHEDLENLTTY GFRGEALGSICCIAEV.LITTR
SwissProt TAADNFSTQYVLDGSGHILSQKPSHLGQGITVTALRLFKNLPVRKQFYSTAKKC
Accession No. KDEIKKIQDLLMSFGILKPDLR1VFVHNKAV1WQKSRVSDHKMALMSVLGTAVM
P54277 NNMESFQYHSEESQTYLSGFLPKCDADHSFTSLSTPERSFIFINSRPVHQKD1LKL1R
Wild type HHYNLKCLICESTRLYPVFFIXIDVPTADVDVNLTPDKSQVLLQNKESVLIALENL
MT.TCYGPLPSTNSYENNKTDVSAADIVLSKTAETDVLFNKVESSGKNYSNVDTS
VIPFQNDMHNDESGICNTDDCLNHQISIGDFGYGHCSSEISNIDICNTICNAFQDISMS
NVSWENSQTEYSKTCFISSVICH.TQSENGNKDHIDESGENEEEAGLENSSEISADE
WSRGN1LKNSVGENIEPVKILVPEKSLPCKVSNNNYPIPEQMNLNEDSCNKKSNVI
DNKSGK VTAYDLLSNRVIKKPMSASALFVQDHRPQFLIENPKTSLEDATLQIEEL
WKTLSEEEICLKYEEKAIICDLERYNSQMKRAIEQESQMSLKDGRKKIKPTSAWN
LAQKHKLKTSLSNQPICLDELLQSQIEKIIRSQNIKMVQ1PFSMICNLKINFKKQNKV
DLEEICDEPCLIHNLRFPDAWLMTSKTEVMLLNPYRVEEALLFKRLLE'NHKLPAEP
LEKPIMLTESLFNGSHYLDVLYKMTADDQRY SGSTYLSDPRLTANGFKIKLIPGV
SITENYLEIEGMANCLPFYGVADLICEILNAILNRNAKEVYECRPRKVISYLEGEAV
RLSRQLPMYLSKEDIQDILYRIVIKHQFGNEIKECVTIGRPITHHLTYLPETT
MIKCLSVEVQAKIRSGLAISSLGQCVEELALNSIDAEAKCVAVRVNMETF'QVQVI 227
Homo sapiens DNGFGMGSDDVEKVGNRYFTSKCHSVQDLENPRFYGFRGEALANIADMASAVE
SwissProt ISSKKNRTMKTFVKLFQSGKALKACEADVTRASAGTTVTVYNLFYQUVRRKC
Accession No. MDPRLF..FEKVRQRIEALSLMHPSISFSLRNDVSGSMVLQLPKTKDVCSRFCQIYGL
Q9UHC I GKSQICLREISFICYKEFELSGYISSF..AHYNKNMQFLFVNKRLVIATKLIIKLIDFLLR
Wild type KESIICKPKNGPTSRQMNSSLRHRSTPELYGlYVINVQCQFCEYDVCMEPAKTLIE
FQNWDTLLFCIQEGVIC.MFLKQEKLFVELSGEDIKEFSEDNGFSLFDATLQKRVTS
DERSN.FQEACNNILDSYEMFNLQSICA'VKRKTTAENVNTQSSRDSEATRKNTNDA
FLYIYESGGPGHSKMTEPSLQNKDSSCSESKMLEQETIVASEAGENEKHKKSFLE
HSSLENPCGTSLEM.FLSPFQTPCHFEESGQDLEIWKESTTVNGMAANILKNNRION
QPKRFKDATEVGCQPLPFATILWGVHSAQT.EKEKKKESSNCGRRNVFSYGRVKL
CSTGFITHVVQNE.K.TKSTETEHSFKNYV.RPG.PTRAQETFGNRTRHSVETPDIKDL
ASTLSKESGQLPNKKNCRTNISYGLENEPTATYTMFSAFQEGSKKSQIDCILSDTS
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PSFPWYRHVSNDSRKTDKLIGFSKPIVRKKL SLSSQLGSLEKFKRQYGK VENPLD
TEVEESNGVTTNLSLQ'VEF'DILLKDKNRLENSDVCKITTMEHSDSDSSCQPASHIL
NSEKFPFSKDEDCLEQQMPSLRESPMTLKELSLFNRKPLDLEKSSESLASKLSRLK
GSERETQTMGIVIMSRFNELPNSDSSRKDSKLCSVLTQDFCMLFNNKHEKTENGVI
PTSDSATQDN SFNKNSKTHSNSNTIENCVISETPL VLPYNNSK VTGKDSDVLIRAS
EQQIGSLDSPSGMLMNPVEDATGDQNGICFQSEESKARACSETEESNTCCSDWQR
HIDVALGRMVYVNKMTGLSTFIAPTEDIQAACTKDLTIVAVDVVLENGSQYRC
QPFRSDLVLPFLPRARAERTVMRQDNRDTVDDTVSSESLQSLFSEWDNPVFARYP
EVAVDVSSGQAESLAVKIHNILYPYRFTKGMMISMQVLQQVDNKFIACLMSTKT
EENGEAGGNLLVLVDQHAAHERIRLEQUIDSYEKQQAQGSGRKKLLSSTLIPPLE
ITVTEEQRRLLWCYHICNLEDLGLEFVFPDTSDSLVLVGKVPLCFVEREANELRRG
RSTVTKSIVEEFIREQLELLQTTGGIQGTLPLTVQKVLASQACHGAIKFNDGLSLQ
ESCRLIEALSSCQLPFQCAHGRPSIVILPLADIDHLEQEKQIKPNLTKLRKMAQAWR
LFGKAECDTRQSLQQSMPPCEPP
MSH2 MAVQPKETLQLESAAEVGFVRFFQGMPEKPITIVRLFDRGDFYTAHGEDALLAA 228
Homo sapiens REVFKTQGVIKYMGPAGAKNLQSV'VLSKMNFESFVKDLILLVRQYRVEVYKNRA
SwissProt GNKASICENDWYLAYKASPGNLSQFEDILFGNNDMSAS1GVVGVKMSAVDGQRQ
Accession No. VGVGYVDS1QRKLGLCEF.PDNDQFSNLEALLIQIGPKECVLPGGETAGDMGKLRQ
P43246 IIQRGGILITERKKADFSTKDIYQDLNRLLKGKKGEQMNSAVLPEIVEENQVAVSSL
Wild type SAVIKFLELLSDDSNFGQFELTTFDFSQY.MKLDIAAVRALNLFQGSVEDTrGSQSL
AALLNKCKTPQGQRLVNQWIKQPLMDICNRIEERLNLVEAFVEDAELRQTLQEDL
LRRFPDLNRLAKKFQRQAANLQDCYRLYQGINQLPNVIQALEKHEGKHQKLLLA
VFVTPLIDLRSDFSKFQEMIETTLDIMDQVENHEFLVKPSFDPNLSELREIMNDLE
KK.MQSTLISAARDLGLDPGKQIKLDSSAQFGYYFRVTCKEEKVLRNNKNFSTVDI
QKNGVKFI'NSKLTSLNEEYTKNKTEYEEAQDALVKEIVNISSGYVEPMQTLNDVL
AQLDAVVSFAHVSNGAPVPY'VRPAJLEKGQGRIILKASRHACVEVQDEIAFIPND
VYFEKDKQMFHIITGPNMGGKSTYIRQTGVIVLMAQIGCFVPCESAEVSIVDCILA
RVGAGDSQLKGVSTFMAEMLETASILRSATKDSLIIIDELGRGTSTYDGFGLAWAI
SEYIATKIGAFCMFATHFHELTALANQIPTVNNLHVTALITEETLTMLYQVKKGV
CDQSFGIHVAELANFPKHVIECAKQKALELEEFQYIGESQGYDIMEPAAKKCYLE
REQGEKIIQEFLSKVKQMPFTEMSEENITIKLKQLKAEVIAKNNSFVNEIISRIKVTI'
MSH6 MSRQSTLYSFFPKSPALSDANK.A.SARASREGGRAAAAPGASPSPGGDAAWSEAG 229
Homo sapiens PGPRPLAR SASPPKAKNLN GGLRRSVAP AAPTSCDFSPGDLVWAKMEGYPWWP
SwissProt CLVYNHPFDG'TFIREKGKSVRVFIVQFFDDSPTRGWVSKRLLKPYTGSKSKEAQK
Accession No. GGHFYSAKPEILRAMQRADEALNKDKIKRLEL AVCDEPSEPEEEEEMEVG1TYV
P52701 TDKSEEDNEIESEEEVQPKTQGSRRSSRQIKKRRVISDSESDIGGSDVEFKPDTKEE
Wild typo GSSDEISSGVGDSESEGLNSPVKVARKRKRMVTGNGSLKRKSSRKETPSATKQAT
SISSETFCNTLRAFSAPQNSESQAHVSGGGDDSSRPTV'VVYHETLEWLICEEKRRDEH
RRRPDHPDFDASTLYVPEDFLNSCTPGMRKWWQIKSQNFDLVICYKVGKFTELY
HMDAL1GVSELGLVFMKGNWAHSGFPEIAF'GRYSDSLVQKGYKVAR'VEQTETPE
MMEARCRKMAHISKYDRVVRREICRIITKGTQTYSVLEGDPSENYSKYLLSLKEK
EEDSSGHTRAYGVCF'VDTSLGKFFIGQFSDDRHCSRFRTLVAHYPPVQVLFEKGN
LSKETICTILKSSLSCSLQEGLIPGSQF'WDASKTLRTLLEEEYFREKLSDGIGVMLPQ
VLICGMTSESDSIGLTPGEKSELALSALGGCVFYLKKCLIDQELLSMANFEEYIPLD
SDTVSTTRSGAIFTKAYQRMVLDAVTLNNLEIFLNGTNGSTEGTLLERVDTCHTP
FGKRLLKQWLCAPLCNHYAINDRLDAIEDLMV'VPDKISEVVELLKKLPDLERLLS
KIHNVGSPLKSQNHPDSRAIMYEETTYSKKKIIDFLSALEGFKVMCKIIGIMEEVA
DGFXSKILKQVISLQTKNPEGRFPDLTVELNRWDTAFDHEKARKTGLITPKAGFD
SDYDQALADIRENEQSLLEYLEKQRNRIGCRTIVYWGIGRNRYQLEIPENFITRNL
PEEYELKSTKKGCKRYWTKTIEKKLANLINAEMRRDVSLKDCMRRLFYNFDKNY
KDWQSAVECIAVLDVLLCLANYSRGGDGPMCRPVILLPEDTPPFLELKGSRHPCI
TKTFFGDDFIPNDILIGCEEEEQE'NGKAYCVLVTGPNMGGKSTLMRQAGLLAVM
AQMGCYVPAEVCRLTPIDRVFTRLGASDRIMSGESTFFVELSETASILMHATAHS
LVLVDELGRGTATFDGTAIANAVVKELAETIKCRTLFSTHYHSLVEDYSQNVAV
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RLGHMACMVENECEDPSQETITFLYKFIKGACPKSYGFNAARLANLPEEVIQKGH
RICAREFEKMNQSLRLFREVCLASERSTVDAEAVHKLLTLIKEL
PCNA. MFEARLVQGSILICICVLEALKDLINEACWDISSSGVNLQSMDSSHVSLVQLTIASE 230
Homo sapiens GFDTYRCDRNLAMGVNLTSMSKILKCAGNEDITTLRAEDNADTLALVFEAPNQE
SwissProt KVSDYEMKLMDLDVEQLGIPEQEYSCVVKMPSGEFARICRDLSHIGDAVVISCA
Accession No. KDGVKFSASGELGNGNIKLSQTSNVDICEEEAVTIEMNEPVQLTFALRYLNFFTKA
P12004 TPLSSTVTLSMSADVPLVVEYKIADMGHLICYYLAPKIEDEEGS
Wild type
RFC MDIRKFFGVIPSGKKINSETVKKNEKTKSDEETLKAKKGIKEIKVNSSRKEDDFK 231
Homo sapiens QKQPSKKKRHYDSDSESEETLQVKNAKKPPEKLPVSSKPGKISRQDPVTYISETDE
SwissProt EDDFMCKKAASKSKENGRSTNSILLGTSNMKKNEENTKTIC.NKPLSPIKLTPTSVL
Accession No. DYFGTGSVQRSNI(KMVASKRKELSQN1DESGLNDEAIAICQLQLDEDAELERQL
P35251 HEDEEFARTLAMLDEEPKTKKARKDTEAGETFSSVQANLSKAEKIIKYPHKVICT
Wild type AQVSDERKSYSPRICQSKYESSKESQQHSKSSADKIGEVSSPICASSKLAINIKRKEE
SSYKEIEPVASKRKENAIKLKGETKTPKKTKSSP.AKKESVSPEDSEKKRTNYQAY
RSYLNREGPKALGSKEIPKGAENCLEGLIFVITGVLESIERDEAKSLIERYGGKVTG
NVSKKTNYLVMGRDSGQSKSDKAAALGTIUIDEDGLLNLIRTMPGICKSICYEIAV
ETEMKKESKLERIPQKNVQGKRKISPSKKESESKK.SRPTSKRDSLAKTIKKEIDV
FWKSLDFKEQVAEETSGDSKARNLADDSSENKVENLLWVDKYKPTSLKTIIGQQ
GDQSCANKLLRWLRNVVQKSSSEDKKHAAICFGKFSGKDDGSSFKAALLSGPPGV
GK1TTASLVMELGYSYVF1.,NASDTRSKSSIKAIVAESLNNTSIKGFYSNGAASS
VSTKHALIMDEVDGMAGNEDRGGIQELIGLIKHTKIPIICMCNDRNHPKIRSLVHY
CFDLRFQRPRVEQ1KGAMMSIAFKEGLICIPPPAMNEHLGANQDIRQVLIINLSMW
CARSKALTYDQAKADSHRAKICDIKMGPFDVARKVFAAGEETAHMSLVDKSDLF
FFIDYSIAPLFVQENYINVKPVAAGGDMKKHI,MLLSRAADSICDGDLVDSQIRSK
QNWSLIPAQAIYASVLPGFLMRGYMTQFPTFPSWLGKHSSTGKHDRIVQDLALH
MSLRTYSSKRTVNMDYLSLIRDALVQPLTSQGVDGVQDVVALMD'TYYLMKFD
FENTMEISSWGGKPSPFSKLDPKVICAAFTRAYNKEAHLTPYSLQAIICASRHSTSPS
LDSEYNEELNF,DDSQSDEKDQDAIETDAMEKKKTKSSKPSKPEKDKEPRICGKGK
SSICK
EX01 MGIQGLLQFIICEASEPIHVRKYKGQVVAVDTYCWLHKGAIACAEKLAICGEF'TDR 156
Homo sapiens YVGFCMKFVNIvILLSHGIKPILVFDCrCTLPSKKEVERSRRERRQANLLKGKQLLR
SwissProt EGKVSEARECFIRSINITHAMAHKVIKAARSQGVDCLVAPYEADAQLAYLNKAG
Accession No. IVQAIITEDSDLLAFGCKKVILKMDQFGNGLEIDQARLGMCRQLGDVFTEEICFRY
Q9UQ84 MCILSGCDYLSSLRGIGLAKACKVLRLANNPDIVKVIKKIGHYLKMNITVPEDYIN
Wild type GFIRANNTFLYQLVFDPEKRKLIPLNAYEDDVDPETLSYAGQYVDDSIALQIALGN
KDINTFEQ1DDYNPDTAMPAHSRSIISWDDKTCQICSANVSSIWHRNYSPRPESGT
VSDAPQLKENPSTVGVERVISTKGLNLPRKSSIVKRPRSAELSEDDLLSQYSLSFT
KKTKICNSSEGNKSLSFSEVFVPDLVNGPTNKKSVSTPPRTRNKFATFLQRKNEES
GAVVVF'GTRSRFFCSSDSTDCVSNKVSIQPLDETAVTDKENNLFIESEYGDQEGICR
LVDTDVARNSSDDIPNNHIPGDHIPDKATWIDEESYSFESSKFTRTISPPTLGTLR
SCFSWSGGLGDFSRTPSPSPSTALQQFRRKSDSPTSLPENN. MSDVSQLKSEESSDD
ESHPLREEACSSQSQESGEFSLQSSNASKLSQCSSKDSDSEESDCNIKLLDSQSDQT
SKLRLSHFSICICDTPLRNICVPGLYKSSSADSLSTTKIKPLGPARASGLSKKPASIQK
RKIIHNAENKPGLQIKLNELWKNFGFKKDSEKLPPCKKPLSPVRDNIQLTPEAEED
1FNKPECGRVQRAIFQ
POL8 MDGKRRPGPGPGVPPKRARGGLWDDDDAPRPSQFEEDLALMEEMEAEHRLQEQ 232
Homo sapiens EEEELQS VLEGVADGQVPPSAIDPRWLRPTPPALDPQMPLIFQQLEIDHYVGPAQ
SwissProt PVPGGPPPSRGSVPVLRAFG'VTDEGFSVCCHIHGFAPYFYTPAPPGFGPEHlvIGDL
Accession No. QRELNLAISRDSRGGRELTGPAVLAVELCSRESMFGYHGHGPSPFLRITVALPRLV
P28340 APARI2LLEQGIRVAGLGTPSFAPYEANVDFEIRFMVDTDIVGCNWLELPAGKYAL
Wild type RLKEXATQCQLEADVLWSD VVSHPPEGPWQR1APLRVLSFDIECAGRKGIFPEPE
RDPVIQICSLGLRWGEPEPFLRLALTLRPCAPILGAKVQSYEKEEDLLQAWSTFIRI
MDPDVITGYNIQNFDLPYLISRAQTLKVQTFPFLGRVAGLCSNIRDSSFQSKQTGR
RDTIKVVSMVGRVQMDMLQVLLREYKLRSYTLNAVSFHFLGEQKEDVQHSIIID
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LQNGNDQTRRRLA VY CLKDAYLPLRLLERLMVLVNA'VEMAR VTGVPLS YLL SR
GQQVKVVSQLLRQAMMEGLLMPVVKSEGGEDYTGATVIEPLKG YYDVP1ATLD
FSSLYPSIMMAHNLC'Y1TLLRPGTAQKLGLTEDQFIRTPTGDEFVKTSVRKGLLP
QILENLLSARKRAKAELAKETDPLRRQVLDGRQLALKVSANSVYGFTGAQVGKL
PCLEISQSVTGFGRQM1EKTKQLVESKYTVEN GYSTSAKVVYGDTDSVMCRFGV
SSVAEAMALGREAADWVSGHFPSP1RLEF'EKVYFPYLLISKKRYAGLLFSSRPDA
HDRMDCKGLEAVRRDNCPL VANLVFASLRRLLIDRDPEGAVAHAQDVISDLLCN
RIDISQLVITKELTRAASDYAGKQAHVELAERMRKRDPGSAPSLGDRVPYVIISAA
KGVAAYMKSEDPLFVLEHSLPIDTQYYLEQQLAKPLLRIFEPILGEGRAEA'VLLR
GDHTRCKTVLTGKVGGLLAFAKRRNCCIGCRTVLSHQGAVCEFCQPRESELYQK
EVSHLNALEERFSRLWTQCQRCQGSLHEDVICTSRDCPIFYMRICKVRKDLEDQE
QLLRRFGPPGPEAW
12981 Thus, in one aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating the DNA mismatch repair (MMR) system.
[299] In another aspect, the present disclosure provides a method for editing
a nucleotide
molecule (e.g., a genome), comprising contacting a target nucleotide molecule
with a prime
editor and an inhibitor of the MMR system, e.g., an inhibitor of one or more
of MLH1, PMS2 (or
MutL alpha), PMS1 (or MutL beta), MLH3 (or Mud, gamma), MutS alpha (MSH2-
MSH6),
MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLS, or PCNA. In various
embodiments, the inhibitor can be a small molecule inhibitor. In other
embodiments, the
inhibitor can be an antibody, e.g., a neutralizing antibody. In still other
embodiments, the
inhibitor can be a dominant negative mutant of one or more of MLHI, PMS2 (or
MutL alpha),
PMS1 (or MutL beta), MLH3 (or MutL gamma), MutS alpha (MSH2-MSH6), MutS beta
(MSH2-MSH3), MSH2, MSH6, PCNA, RFC, EX01, POLS, or PCNA, e.g., a dominant
negative
mutant of MLH1. In still other embodiments, the inhibitor can be targeted at
the level of
transcription, e.g., an siRNA or other nucleic acid agent that knocks down the
level of a
transcript encoding MLH1, PMS2 (or MutL alpha), PMS I (or MutL beta), MLFL3
(or MutL
gamma), MutS alpha (MSH2-MSH6), MutS beta (MSH2-MSH3), MSH2, MSH6, PCNA, RFC,
EX01, POLE, or PCNA. In yet other embodiments, the step of "contacting a
target nucleotide
molecule with a prime editor" can include (i) delivering directly to a cell an
effective amount of
a prime editor fusion protein (e.g., PE1 or PE2) complexed with a lipid
delivery system; (ii)
delivery to a cell a mRNA or delivery complex comprising an inRNA that encodes
a prime editor
fusion protein and/or a suitable pegRNA; and (iii) a DNA vector (e.g., an AAV
or lentivirus
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vector, plasmid, or other nucleic acid delivery vector) that encodes a prime
editor fusion protein
and/or a suitable pegRNA on one or more DNA vectors.
[300] In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating MLH1 or variant thereof. In another aspect, the present
disclosure provides a
method for editing a nucleotide molecule (e.g., a genome), comprising
contacting a target
nucleotide molecule with a prime editor and an inhibitor of MLH1. In various
embodiments, the
inhibitor can be a small molecule inhibitor. In other embodiments, the
inhibitor can be an anti-
MLH1 antibody, e.g., a neutralizing antibody that inactivates MLH1. In still
other embodiments,
the inhibitor can be a dominant negative mutant of MLH1. In still other
embodiments, the
inhibitor can be targeted at the level of transcription of MLH1, e.g., an
siRNA or other nucleic
acid agent that knocks down the level of a transcript encoding MLH1. In yet
other embodiments,
the step of "contacting a target nucleotide molecule with a prime editor" can
include (i)
delivering directly to a cell an effective amount of a prime editor fusion
protein (e.g., PE1 or
PE2) complexed with a lipid delivery system; (ii) delivery to a cell a MRNA or
delivery complex
comprising an mRNA that encodes a prime editor fusion protein and/or a
suitable pegRNA; and
(iii) a DNA vector (e.g., an AA.V or lentivirus vector, plasmid, or other
nucleic acid delivery
vector) that encodes a prime editor fusion protein and/or a suitable pegRNA on
one or more
DNA vectors.
[301] In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating PMS2 (or MutL alpha) or variant thereof. In another aspect, the
present disclosure
provides a method for editing a nucleotide molecule (e.g., a genome),
comprising contacting a
target nucleotide molecule with a prime editor and an inhibitor of PMS2 (or
MutL alpha). In
various embodiments, the inhibitor can be a small molecule inhibitor. In other
embodiments, the
inhibitor can be an anti-PMS2 (or MutL alpha) antibody, e.g., a neutralizing
antibody that
inactivates PMS2 (or MutL alpha). In still other embodiments, the inhibitor
can be a dominant
negative mutant of PMS2 (or MutL alpha). In still other embodiments, the
inhibitor can be
targeted at the level of transcription of PMS2 (or MutL alpha), e.g., an siRNA
or other nucleic
acid agent that knocks down the level of a transcript encoding ML PMS2 (or
MutL alpha). In yet
other embodiments, the step of "contacting a target nucleotide molecule with a
prime editor" can
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include (i) delivering directly to a cell an effective amount of a prime
editor fusion protein (e.g.,
PEI or PE2) cornplexed with a lipid delivery system; (ii) delivery to a cell a
mRNA or delivery
complex comprising an mRNA that encodes a prime editor fusion protein and/or a
suitable
pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector, plasmid, or
other nucleic
acid delivery vector) that encodes a prime editor fusion protein and/or a
suitable pegRNA on one
or more DNA vectors.
[302] In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating PMS1 (or MutL beta) or variant thereof. In another aspect, the
present disclosure
provides a method for editing a nucleotide molecule (e.g., a genome),
comprising contacting a
target nucleotide molecule with a prime editor and an inhibitor of PMS1 (or
MutL beta). In
various embodiments, the inhibitor can be a small molecule inhibitor. In other
embodiments, the
inhibitor can be an anti-PMS1 (or MutL beta) antibody, e.g., a neutralizing
antibody that
inactivates PMS1 (or MutL beta). In still other embodiments, the inhibitor can
be a dominant
negative mutant of PMS1 (or MutL beta). In still other embodiments, the
inhibitor can be
targeted at the level of transcription of PMS1 (or MutL beta), e.g., an siRNA
or other nucleic
acid agent that knocks down the level of a transcript encoding PMS1 (or MutL
beta). In yet other
embodiments, the step of "contacting a target nucleotide molecule with a prime
editor" can
include (i) delivering directly to a cell an effective amount of a prime
editor fusion protein (e.g.,
PEI or PE2) complexed with a lipid delivery system; (ii) delivery to a cell a
mRNA or delivery
complex comprising an mRNA that encodes a prime editor fusion protein and/or a
suitable
pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivinis vector, plasmid, or
other nucleic
acid delivery vector) that encodes a prime editor fusion protein and/or a
suitable pegRNA on one
or more DNA vectors.
[303] In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating MLH3 (or MutL gamma) or variant thereof. In another aspect, the
present disclosure
provides a method for editing a nucleotide molecule (e.g., a genome),
comprising contacting a
target nucleotide molecule with a prime editor and an inhibitor of MLH3 (or
MutL gamma). In
various embodiments, the inhibitor can be a small molecule inhibitor. In other
embodiments, the
inhibitor can be an anti-MLH3 (or MutL gamma) antibody, e.g., a neutralizing
antibody that
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inactivates MLH3 (or MutL gamma). In still other embodiments, the inhibitor
can be a dominant
negative mutant of MLH3 (or MutL gamma). In still other embodiments, the
inhibitor can be
targeted at the level of transcription of P MLH3 (or Mud, gamma), e.g., an
siRNA or other
nucleic acid agent that knocks down the level of a transcript encoding MLH3
(or MutL gamma).
In yet other embodiments, the step of "contacting a target nucleotide molecule
with a prime
editor" can include (i) delivering directly to a cell an effective amount of a
prime editor fusion
protein (e.g., PE1 or PE2) complexed with a lipid delivery system; (ii)
delivery to a cell a mRNA
or delivery complex comprising an mRNA that encodes a prime editor fusion
protein and/or a
suitable pegRNA; and (iii) a DNA vector (e.g., an AAV or lentivirus vector,
plasmid, or other
nucleic acid delivery vector) that encodes a prime editor fusion protein
and/or a suitable pegRNA
on one or more DNA vectors.
[304] In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating MutS alpha (MSH2-MSH6) or variant thereof. In another aspect, the
present
disclosure provides a method for editing a nucleotide molecule (e.g., a
genome), comprising
contacting a target nucleotide molecule with a prime editor and an inhibitor
of MutS alpha
(MSH2-MSH6). In various embodiments, the inhibitor can be a small molecule
inhibitor. In
other embodiments, the inhibitor can be an anti-MutS alpha (MSH2-MSH6)
antibody, e.g., a
neutralizing antibody that inactivates MutS alpha (MSH2-MSH6). In still other
embodiments,
the inhibitor can be a dominant negative mutant of MutS alpha (MSH2-MSH6). In
still other
embodiments, the inhibitor can be targeted at the level of transcription of
MutS alpha (MSH2-
MSH6), e.g., an siRNA or other nucleic acid agent that knocks down the level
of a transcript
encoding MutS alpha (MSH2-MSH6). In yet other embodiments, the step of
"contacting a target
nucleotide molecule with a prime editor" can include (i) delivering directly
to a cell an effective
amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a
lipid delivery
system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a
prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector
(e.g., an AAV or
lentivirus vector, plasmid, or other nucleic acid delivery vector) that
encodes a prime editor
fusion protein and/or a suitable pegRNA on one or more DNA vectors.
[305] In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
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inactivating MSH2 or variant thereof. In another aspect, the present
disclosure provides a method
for editing a nucleotide molecule (e.g., a genome), comprising contacting a
target nucleotide
molecule with a prime editor and an inhibitor of MSH2. In various embodiments,
the inhibitor
can be a small molecule inhibitor. In other embodiments, the inhibitor can be
an anti- MSH2
antibody, e.g., a neutralizing antibody that inactivates MSH2. In still other
embodiments, the
inhibitor can be a dominant negative mutant of MSH2. In still other
embodiments, the inhibitor
can be targeted at the level of transcription of MSH2, e.g., an siRNA or other
nucleic acid agent
that knocks down the level of a transcript encoding MSH2. In yet other
embodiments, the step of
"contacting a target nucleotide molecule with a prime editor" can include (i)
delivering directly
to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or
PE2) complexed with
a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex
comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii)
a DNA vector
(e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery
vector) that encodes a
prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
[306] In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating MSH6 or variant thereof. In another aspect, the present
disclosure provides a method
for editing a nucleotide molecule (e.g., a genome), comprising contacting a
target nucleotide
molecule with a prime editor and an inhibitor of MSH6. In various embodiments,
the inhibitor
can be a small molecule inhibitor. In other embodiments, the inhibitor can be
an anti- MSH6
antibody, e.g., a neutralizing antibody that inactivates MSH6. In still other
embodiments, the
inhibitor can be a dominant negative mutant of MSH6. In still other
embodiments, the inhibitor
can be targeted at the level of transcription of MSH6, e.g., an siRNA or other
nucleic acid agent
that knocks down the level of a transcript encoding MSH6. In yet other
embodiments, the step of
"contacting a target nucleotide molecule with a prime editor" can include (i)
delivering directly
to a cell an effective amount of a prime editor fusion protein (e.g., PEI or
PE2) complexed with
a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex
comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii)
a DNA vector
(e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery
vector) that encodes a
prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
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[307] In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating PCNA or variant thereof. In another aspect, the present
disclosure provides a method
for editing a nucleotide molecule (e.g., a genome), comprising contacting a
target nucleotide
molecule with a prime editor and an inhibitor of PCNA. In various embodiments,
the inhibitor
can be a small molecule inhibitor. In other embodiments, the inhibitor can be
an anti- PCNA
antibody, e.g., a neutralizing antibody that inactivates PCNA. In still other
embodiments, the
inhibitor can be a dominant negative mutant of PCNA. In still other
embodiments, the inhibitor
can be targeted at the level of transcription of PCNA, e.g., an siRNA or other
nucleic acid agent
that knocks down the level of a transcript encoding PCNA. In yet other
embodiments, the step of
"contacting a target nucleotide molecule with a prime editor" can include (i)
delivering directly
to a cell an effective amount of a prime editor fusion protein (e.g., PE1 or
PE2) complexed with
a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex
comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii)
a DNA vector
(e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery
vector) that encodes a
prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
[308] In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating RFC or variant thereof. In another aspect, the present disclosure
provides a method
for editing a nucleotide molecule (e.g., a genome), comprising contacting a
target nucleotide
molecule with a prime editor and an inhibitor of RFC. In various embodiments,
the inhibitor can
be a small molecule inhibitor. In other embodiments, the inhibitor can be an
anti-RFC antibody,
e.g., a neutralizing antibody that inactivates RFC. In still other
embodiments, the inhibitor can be
a dominant negative mutant of IRFC. In still other embodiments, the inhibitor
can be targeted at
the level of transcription of RFC, e.g., an siRNA or other nucleic acid agent
that knocks down
the level of a transcript encoding RFC. In yet other embodiments, the step of
"contacting a target
nucleotide molecule with a prime editor" can include (i) delivering directly
to a cell an effective
amount of a prime editor fusion protein (e.g., PE1 or PE2) complexed with a
lipid delivery
system; (ii) delivery to a cell a mRNA or delivery complex comprising an mRNA
that encodes a
prime editor fusion protein and/or a suitable pegRNA; and (iii) a DNA vector
(e.g., an AAV or
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lentivirus vector, plasmid, or other nucleic acid delivery vector) that
encodes a prime editor
fusion protein and/or a suitable pegRNA on one or more DNA vectors.
[309] In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating EXO I or variant thereof. In another aspect, the present
disclosure provides a method
for editing a nucleotide molecule (e.g., a genome), comprising contacting a
target nucleotide
molecule with a prime editor and an inhibitor of EX01. In various embodiments,
the inhibitor
can be a small molecule inhibitor. In other embodiments, the inhibitor can be
an anti- EX01
antibody, e.g., a neutralizing antibody that inactivates EX01. In still other
embodiments, the
inhibitor can be a dominant negative mutant of EX01. In still other
embodiments, the inhibitor
can be targeted at the level of transcription of EX01, e.g., an siRNA or other
nucleic acid agent
that knocks down the level of a transcript encoding EX0I. In yet other
embodiments, the step of
"contacting a target nucleotide molecule with a prime editor" can include (1)
delivering directly
to a cell an effective amount of a prime editor fusion protein (e.g., PEI or
PE2) complexed with
a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex
comprising an mRNA
that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii)
a DNA vector
(e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery
vector) that encodes a
prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
[310] In still another aspect, the present disclosure provides a method for
editing a nucleotide
molecule (e.g., a genome) using prime editing while blocking, inhibiting, or
otherwise
inactivating POLS or variant thereof. In another aspect, the present
disclosure provides a method
for editing a nucleotide molecule (e.g., a genome), comprising contacting a
target nucleotide
molecule with a prime editor and an inhibitor of POLO. In various embodiments,
the inhibitor
can be a small molecule inhibitor. In other embodiments, the inhibitor can be
an anti- POLO
antibody, e.g., a neutralizing antibody that inactivates POLS. In still other
embodiments, the
inhibitor can be a dominant negative mutant of POLO. In still other
embodiments, the inhibitor
can be targeted at the level of transcription of POLO, e.g., an siRNA or other
nucleic acid agent
that knocks down the level of a transcript encoding POLO. In yet other
embodiments, the step of
"contacting a target nucleotide molecule with a prime editor" can include (i)
delivering directly
to a cell an effective amount of a prime editor fusion protein (e.g., PEI. or
PE2) complexed with
a lipid delivery system; (ii) delivery to a cell a mRNA or delivery complex
comprising an mRNA
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that encodes a prime editor fusion protein and/or a suitable pegRNA; and (iii)
a DNA vector
(e.g., an AAV or lentivirus vector, plasmid, or other nucleic acid delivery
vector) that encodes a
prime editor fusion protein and/or a suitable pegRNA on one or more DNA
vectors.
13111 In still other aspects, the present disclosure provides methods for
prime editing whereby
correction by the MMR pathway of the alterations introduced into a target
nucleic acid molecule
is evaded, without the need to provide an inhibitor of the MMR pathway.
Surprisingly, pegRNAs
designed with consecutive nucleotide mismatches compared to a target site on
the target nucleic
acid, for example, pegRNAs that have three or more consecutive mismatching
nucleotides, can
evade correction by the MMR pathway, resulting in an increase in prime editing
efficiency
and/or a decrease in the frequency of indel formation compared to the
introduction of a single
nucleotide mismatch using prime editing. In addition, insertions and deletions
of multiple
consecutive nucleotides, for example, three or more contiguous nucleotides, or
10 or more
contiguous nucleotides in length introduced by prime editing may also evade
correction by the
MMR pathway, resulting in an increase in prime editing efficiency and/or a
decrease in the
frequency of indel formation compared to prime editing with a corresponding
control pegRNA
(e.g., a control pegRNA that does not introduce insertion or deletion of three
or more contiguous
nucleotides). In some embodiments, prime editing that introduces insertion or
deletion of 10 or
more contiguous nucleotides results in an increase in prime editing efficiency
and/or a decrease
in indel frequency compared to the introduction of an insertion or deletion of
less than 10
nucleotides in length using prime editing.
[312] Thus, in one aspect, the present disclosure provides methods for editing
a nucleic acid
molecule by prime editing comprising contacting a nucleic acid molecule with a
prime editor and
a pegRNA comprising a DNA synthesis template on its extension arm comprising
three or more
consecutive nucleotide mismatches relative to a target site on the nucleic
acid molecule. In some
embodiments, the pegRNA comprises a DNA synthesis template comprising one or
more
nucleotide edits compared to the endogenous sequence of the nucleic acid
molecule (e.g., a
double stranded target DNA) to be edited, wherein the one or more nucleotide
edits comprises (i)
an intended change is an insertion, deletion, or substitution of x consecutive
nucleotides that
corrects a mutation (e.g. a disease associated mutation) in the nucleic acid
molecule, and (ii) an
insertion, deletion, or substitution of y consecutive nucleotides directly
adjacent to the x
nucleotides, wherein (x+y) is an integer no less than 3. In some embodiments,
the insertion,
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deletion, or substitution of the y consecutive nucleotides is a silent
mutation. In some
embodiments, the insertion, deletion, or substitution of the y consecutive
nucleotides is a benign
mutation. The silent mutations may be present in coding regions of the target
nucleic acid
molecule or in non-coding regions of the target nucleic acid molecule. When
the silent mutations
are present in a coding region, they introduce into the nucleic acid molecule
one or more
alternate codons encoding the same amino acid as the unedited nucleic acid
molecule.
Alternatively, when the silent mutations are in a non-coding region or a
junction of a coding
region and a non-coding region (e.g., an intron/exon junction), the silent
mutations may be
present in a region of the nucleic acid molecule that does not influence
splicing, gene regulation,
RNA lifetime, or other biological properties of the target site on the nucleic
acid molecule. A.
benign mutation may refer to a nucleotide alteration or amino acid alteration
that alters the amino
acid sequence of the protein or polypeptide encoded by the target nucleic acid
sequence, but does
not impair or substantively impair expression and/or function of the protein
or polypeptide. In
some embodiments, x is an integer between 1 and 50. In some embodiments, y is
an integer
between 1 and 50. In some embodiments, y is an integer no less than 1. In some
embodiments,
the inclusion of the silent mutation(s) increases the efficiency, reduces
unintended indel
frequency, and/or improves editing outcome purity by prime editing. As used
herein, the term
"prime editing outcome purity" may refer to the ratio of intended edit to
unintended indels that
result from prime editing. In some embodiments, the inclusion of the silent
mutation(s) increases
the efficiency, reduces unintended indel frequency, and/or improves editing
outcome purity by
prime editing by at least 1.5-fold, at least 2.0 fold, at least 2.5-fold, at
least 3.0-fold, at least 3.5-
fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 5.5-
fold, at least 6.0-fold, at least
6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least
8.5-fold, at least 9.0-fold, at
least 9.5-fold, at least 10.0 fold, at least 11-fold, at least 12-fold, at
least 13-fold, at least 14-fold,
at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at
least 19-fold, at least 20-fold,
at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at
least 25-fold, at least 26-fold,
at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at
least 31-fold, at least 32-fold,
at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at
least 37-fold, at least 38-fold,
at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at
least 43-fold, at least 44-fold,
at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at
least 49-fold, at least 50-fold,
at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at
least 55-fold, at least 56-fold,
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at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at
least 61-fold, at least 62-fold,
at least 63-fold, at least 64-fold, at least 65-fold, at least 66-fold,at
least 67-fold, at least 68-fold,
at least 69-fold, at least 70-fold, at least 71-fold, at least 72-fold, at
least 73-fold, at least 74-fold,
or at least 75-fold compared to prime editing with a control pegRNA that does
not include the
silent mutation(s), e.g., a control pegRNA that only includes the insertion,
deletion, or
substitution of the x consecutive nucleotides and not the insertion, deletion,
or substitution of the
y consecutive nucleotides.
13131 In some embodiments, at least one of the three or more consecutive
nucleotide
mismatches results in an alteration in the amino acid sequence of a protein
expressed from the
nucleic acid molecule. In some embodiments, more than one of the consecutive
nucleotide
mismatches results in an alteration in the amino acid sequence of a protein
expressed from the
nucleic acid molecule. In some embodiments, at least one of the nucleotide
mismatches are silent
mutations that do not result in an alteration in the amino acid sequence of a
protein expressed
from the nucleic acid molecule. The silent mutations may be present in coding
regions of the
target nucleic acid molecule or in non-coding regions of the target nucleic
acid molecule. When
the silent mutations are present in a coding region, they introduce into the
nucleic acid molecule
one or more alternate codons encoding the same amino acid as the unedited
nucleic acid
molecule. Alternatively, when the silent mutations are in a non-coding region,
the silent
mutations may be present in a region of the nucleic acid molecule that does
not influence
splicing, gene regulation, RNA lifetime, or other biological properties of the
target site on the
nucleic acid molecule.
[314] Any number of consecutive nucleotide mismatches of three or more can be
used to
achieve the benefits of evading correction by the MMR pathway. In some
embodiments, the
DNA synthesis template of the extension arm on the pegRNA comprises 3, 4, 5,
6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive
nucleotide mismatches
relative to the endogenous sequence of a target site in the nucleic acid
molecule edited by prime
editing. In some embodiments, the DNA synthesis template of the extension arm
on the pegRNA
comprises 3, 4, or 5 consecutive nucleotide mismatches relative to the
endogenous sequence of a
target site in the nucleic acid molecule edited by prime editing. In some
embodiments, the DNA
synthesis template of the extension arm on the pegRNA comprises 6, 7, 8, 9,
10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 consecutive nucleotide mismatches relative to the
endogenous sequence
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of a target site in the nucleic acid molecule edited by prime editing. In some
embodiments, the
DNA synthesis template of the extension arm on the pegRNA comprises three or
more, four or
more, five or more, six or more, seven or more, eight or more, nine or more,
or ten or more
consecutive nucleotide mismatches relative to a target site on the nucleic
acid molecule.
[315] In another aspect, the present disclosure provides methods for editing a
nucleic acid
molecule by prime editing comprising contacting a nucleic acid molecule with a
prime editor and
a pegRNA comprising a DNA synthesis template on its extension arm comprising
an insertion or
deletion of 10 or more nucleotides relative to a target site on the nucleic
acid molecule.
Insertions and deletions of 10 or more nucleotides in length evade correction
by the MMR
pathway when introduced by prime editing and thus can benefit from the
inhibition of the MMR.
pathway without the need to provide an inhibitor of MMR. Insertions and
deletions of any length
greater than 10 nucleotides can be used to achieve the benefits of evading
correction by the
MMR pathway. lin some embodiments, the DNA synthesis template comprises an
insertion or
deletion of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25
nucleotides relative to
the endogenous sequence at a target site of the nucleic acid molecule edited
by prime editing. In
some embodiments, the DNA synthesis template comprises an insertion or
deletion of 11 or
more nucleotides, 12 or more nucleotides, 13 or more nucleotides, 14 or more
nucleotides, 15 or
more nucleotides, 16 or more nucleotides, 17 or more nucleotides, 18 or more
nucleotides, 19 or
more nucleotides, 20 or more nucleotides, 21 or more nucleotides, 22 or more
nucleotides, 23 or
more nucleotides, 24 or more nucleotides, or 25 or more nucleotides relative
to a target site on a
nucleic acid molecule. In certain embodiments, the DNA synthesis template
comprises an
insertion or deletion of 15 or more nucleotides relative to a target site on
the nucleic acid
molecule.
[316] The present disclosure provides compositions and methods for prime
editing with
improved editing efficiency and/or reduced indel formation by inhibiting the
DNA mismatch
repair pathway while conducting prime editing of a target site. Accordingly,
the present
disclosure provides a method for editing a nucleic acid molecule by prime
editing that involves
contacting a nucleic acid molecule with a prime editor, a pegRNA, and an
inhibitor of the DNA
mismatch repair pathway, thereby installing one or more modifications to the
nucleic acid
molecule at a target site with increased editing efficiency and/or lower indel
formation. The
present disclosure further provides polynucleotides for editing a DNA target
site by prime
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editing comprising a nucleic acid sequence encoding a napDNAbp, a polymerase,
and an
inhibitor of the DNA mismatch repair pathway, wherein the napDNAbp and
polymerase is
capable in the presence of a pegRNA of installing one or more modifications in
the DNA target
site with increased editing efficiency and/or lower indel formation. Thus, the
methods and
compositions described herein utilize prime editors, which may comprise a
nucleic acid
programmable DNA binding protein (napDNAbp).
Prime editors: napDNAbp domain
[31 7] In one aspect, a napDNAbp of the prime editors described herein can be
associated with
or complexed with at least one vide nucleic acid (e.g., guide RNA or a
PEgRNA), which
localizes the napDNAbp to a DNA sequence that comprises a DNA strand (i.e., a
target strand)
that is complementary to the guide nucleic acid, or a portion thereof (e.g.,
the spacer of a guide
RNA which anneals to the protospacer of the DNA target). In other words, the
guide nucleic-acid
"programs" the napDNAbp (e.g., Cas9 or equivalent) to localize and bind to
complementary
sequence of the protospacer in the DNA.
[318] Any suitable napDNAbp may be used in the prime editors utilized in the
methods and
compositions described herein. In various embodiments, the napDNAbp may be any
Class 2
CRISPR-Cas system, including any type H, type V. or type VI CRISPR-Cas enzyme.
Given the
rapid development of CRISPR-Cas as a tool for genome editing, there have been
constant
developments in the nomenclature used to describe and/or identify CRISPR-Cas
enzymes, such
as Cas9 and Cas9 orthologs. This application references CRISPR-Cas enzymes
with
nomenclature that may be old and/or new. The skilled person will be able to
identify the specific
CRISPR-Cas enzyme being referenced in this Application based on the
nomenclature that is
used, whether it is old (i.e., "legacy") or new nomenclature. CRISPR-Cas
nomenclature is
extensively discussed in Makarova et aL, "Classification and Nomenclature of
CRISPR-Cas
Systems: Where from Here?," The CRISPR Journal, Vol. 1. No. 5, 2018, the
entire contents of
which are incorporated herein by reference. The particular CRISPR-Cas
nomenclature used in
any given instance in this Application is not limiting in any way and the
skilled person will be
able to identify which CRISPR-Cas enzyme is being referenced.
[319] For example, the following type 11, type V. and type VI Class 2 CRISPR-
Cas enzymes
have the following art-recognized old (i.e., legacy) and new names. Each of
these enzymes,
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and/or variants thereof, may be used with the prime editors utilized in the
methods and
compositions described herein:
Legacy nomenclature J Cu ne u eu datti re4
type II CRISPR-Cas enzymes
Cas9
type V CRISPR-Cas enzymes
Cpfl Cas12a
CasX Cas 12e __
C2c 1 Cas12b1
Cas 12b2 _____________________________ same
C2c3 Cas 12c
CasY Cas12d
C2c4 same
C2c8 same
C2c5 same
C2c10 same
C2c9 same
type 1,7 CRISPR-Cas enzymes
C2c2 Cas 13a
Cas 13d same
C2c7 Cas 13c
C2c6 Cas13b
* See Makarova et aL, The CRISPR Journal, Vol. 1, No. 5, 2018
[320] Without being bound by theory, the mechanism of action of certain
napDNAbp
contemplated herein includes the step of forming an R-loop whereby the
napDNAbp induces the
unwinding of a double-strand DNA target, thereby separating the strands in the
region bound by
the napDNAbp. The guide RNA spacer then hybridizes to the "target strand" at a
region that is
complementary to the protospacer sequence. This displaces a "non-target
strand" that is
complementary to the target strand, which forms the single strand region of
the R-loop. In some
embodiments, the napDNAbp includes one or more nuclease activities, which then
cut the DNA
leaving various types of lesions. For example, the napDNAbp may comprises a
nuclease activity
that cuts the non-target strand at a first location, and/ or cuts the target
strand at a second
location. Depending on the nuclease activity, the target DNA can be cut to
form a "double-
stranded break" whereby both strands are cut. In other embodiments, the target
DNA can be cut
at only a single site, i.e., the DNA is "nicked" on one strand. Exemplary
napDNAbp with
different nuclease activities include "Cas9 nickase" ("nCas9") and a
deactivated Cas9 having no
nuclease activities ("dead Cas9" or "dCas9").
[321] The below description of various napDNAbps which can be used in
connection with the
prime editors utilized in the presently disclosed methods and compositions is
not meant to be
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limiting in any way. The prime editors may comprise the canonical SpCas9, or
any ortholog
Cas9 protein, or any variant Cas9 protein ¨including any naturally occurring
variant, mutant, or
otherwise engineered version of Cas9 ¨ that is known or that can be made or
evolved through a
directed evolutionary or otherwise rnutagenic process. In various embodiments,
the Cas9 or Cas9
variants have a nickase activity, i.e., only cleave one strand of the target
DNA sequence. In other
embodiments, the Cas9 or Cas9 variants have inactive nucleases, i.e., are
"dead" Cas9 proteins.
Other variant Cas9 proteins that may be used are those having a smaller
molecular weight than
the canonical SpCas9 (e.g., for easier delivery) or having modified or
rearranged primary amino
acid structure (e.g., the circular permutant formats).
[322] The prime editors utilized in the methods and compositions described
herein may also
comprise Cas9 equivalents, including Cas12a (Cpfl) and Cas12b1 proteins which
are the result
of convergent evolution. The napDNAbps used herein (e.g., SpCas9, Cas9
variant, or Cas9
equivalents) may also contain various modifications that alter/enhance their
PAM specificities.
Lastly, the application contemplates any Cas9, Cas9 variant, or Cas9
equivalent which has at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or at
least 99.9% sequence identity to a reference Cas9 sequence, such as a
reference SpCas9
canonical sequence or a reference Cas9 equivalent (e.g., Cas12a (Cpfl)).
[323] The napDNAbp can be a CRISPR (clustered regularly interspaced short
palindromic
repeat)-associated nuclease. As outlined above, CRISPR is an adaptive immune
system that
provides protection against mobile genetic elements (viruses, transposable
elements, and
conjugative plasmids). CRISPR clusters contain spacers, sequences
complementary to
antecedent mobile elements, and target invading nucleic acids. CRISPR clusters
are transcribed
and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct
processing of
pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous
ribonuclease 3 (rnc),
and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided
processing of pre-
crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves a linear
or circular
dsDNA target complementary to the spacer. The target strand not complementary
to crRNA is
first cut endonucleolytically, then trimmed 3"-5' exonucleolytically. In
nature, DNA-binding and
cleavage typically requires protein and both RNAs. However, single guide RNAs
("sgRNA", or
simply "gRNA") can be engineered so as to incorporate aspects of both the
crRNA and
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