Note: Descriptions are shown in the official language in which they were submitted.
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INIR12 TRANSGENIC MAIZE
REFERENCE TO SEQUENCE LISTING SUBMITTED
ELECTRONICALLY
100011 The sequence listing contained in the file named "10085W01 ST25.txt",
which was
created on July 28, 2021 and electronically filed on July 28, 2021, is
incorporated herein by
reference in its entirety.
BACKGROUND
[0002] Transgenes which are placed into different positions in the plant
genome through non-site
specific integration can exhibit different levels of expression (Weising et
al., 1988, Ann. Rev.
Genet. 22:421-477). Such transgene insertion sites can also contain various
undesirable
rearrangements of the foreign DNA elements that include deletions and/or
duplications.
Furthermore, many transgene insertion sites can also comprise selectable or
scoreable marker
genes which in some instances are no longer required once a transgenic plant
event containing the
linked transgenes which confer desirable traits are selected.
[0003] Commercial transgenic plants typically comprise one or more independent
insertions of
transgenes at specific locations in the host plant genome that have been
selected for features that
include expression of the transgene(s) of interest and the transgene-conferred
trait(s), absence or
minimization of rearrangements, and normal Mendelian transmission of the
trait(s) to progeny. An
example of a selected transgenic maize event which confers tolerance to
certain lepidopteran insect
pests is the MIR162 transgenic maize event disclosed in U.S. Patent No.;
8455720. MIR162
transgenic maize plants express a VIP3Aa20 protein which can confer resistance
to fall armyworm
(Spodoptera frugiperda), corn earworm (Helicoverpa zea), western bean cutworm
(Striacosta
albicosta), and black cutworm (Agrotis ipsilon) infestations. MIR162
transgenic maize plants also
express a phosphomannose isomerase selectable marker protein.
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[0004] Methods for removing selectable marker genes and/or duplicated
transgenes in transgene
insertion sites in plant genomes involving use of site-specific recombinase
systems (e.g., cre-lox)
as well as for insertion of new genes into transgene insertion sites have been
disclosed (Srivastava
and Ow; Methods Mol Biol, 2015,1287:95-103; Dale and Ow, 1991, Proc. Natl
Acad. Sci.
USA 88, 10558-10562; Srivastava and Thomson, Plant Biotechnol J,
2016;14(2):471-82). Such
methods typically require incorporation of the recombination site sequences
recognized by the
recombinase at particular locations within the transgene.
SUMMARY
[0005] Transgenic maize plant cells comprising a first ZmUbiInt promoter, a
vip3Aa19 or
vip3Aa20 coding region which is operably linked to said promoter, a CaMV 35S
terminator
element which is operably linked to said vip3Aa19 or vip3Aa20 coding region,
and a nopaline
synthase terminator element, wherein said cell does not contain a second
ZmUbiInt promoter and
an operably linked phosphomannose isomerase coding region between said
terminator elements
are provided. Transgenic maize plant cell comprising a nucleotide sequence
comprising a first
ZmUbiInt promoter, a vip3Aa19 or vip3Aa20 coding region which is operably
linked to said
promoter, a CaMV 35S terminator element which is operably linked to said
vip3Aa19 or vip3Aa20
coding region, and a nopaline synthase terminator element, wherein said
nucleotide sequence does
not contain a second ZmUbiInt promoter and an operably linked phosphomannose
isomerase
coding region between said terminator elements are provided. Transgenic maize
plant cells
comprising a nucleotide sequence comprising a ZmUbiInt promoter, a vip3Aa19 or
vip3Aa20
coding region which is operably linked to said promoter, a CaMV 35S terminator
element which
is operably linked to said vip3Aa19 or vip3Aa20 coding region, and a nopaline
synthase terminator
element, wherein said nucleotide sequence does not contain a phosphomannose
isomerase coding
region between said terminator elements are provided. Transgenic maize plant
cells comprising a
nucleotide sequence comprising a first ZmUbiInt promoter, a vip3Aa19 or
vip3Aa20 coding region
which is operably linked to said promoter, a CaMV 35S terminator element which
is operably
linked to said vip3Aa19 or vip3Aa20 coding region, and a nopaline synthase
terminator element,
wherein said nucleotide sequence does not contain a second ZmUbiInt promoter
and an operably
linked phosphomannose isomerase coding region are provided. Transgenic maize
plant cell
comprising a nucleotide sequence comprising a ZmUbiInt promoter, a vip3Aa19 or
vip3Aa20
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coding region which is operably linked to said promoter, a CaMV 35S terminator
element which
is operably linked to said vip3Aa19 or vip3Aa20 coding region, and a nopaline
synthase terminator
element, wherein said nucleotide sequence does not contain a phosphomannose
isomerase coding
region are provided. In certain embodiments, aforementioned transgenic maize
plant cells wherein
:(i) the ZmUbiInt promoter, the vip3Aa19 or vip3Aa20 coding region which is
operably linked to
said promoter, the CaMV 35S terminator element which is operably linked to
said vip3Aa19 or
vip3Aa20 coding region are located in the maize plant cell genomic location of
the MIR162
transgenic locus; (ii) wherein a selectable marker or scoreable is absent from
said maize plant cell
genomic location, and/or (iii) wherein the nopaline synthase terminator
element is not separated
from the CaMV 35S terminator element by DNA encoding a selectable marker
protein, a scoreable
marker protein, or a protein conferring a useful trait are provided.
Transgenic maize plant cells
comprising an INIR12 transgenic locus comprising the first ZmUbiInt promoter,
the vip3Aa19 or
vip3Aa20 coding region which is operably linked to said promoter, the CaMV 35S
terminator
element which is operably linked to said vip3Aa19 or vip3Aa20 coding region,
and the nopaline
synthase terminator element of an original MIR162 transgenic locus allelic
variants thereof, or
other variants thereof, wherein DNA of said original MIR162 transgenic locus,
allelic variants
thereof, or other variants thereof comprising a second ZmUbiInt promoter and
an operably linked
phosphomannose isomerase coding region is absent are provided. In certain
embodiments, the
original MIR162 transgenic locus is set forth in SEQ ID NO: 1, is present in
seed deposited at the
ATCC under accession No. PTA-8166 (SEQ ID NO: 46) or progeny thereof, is an
allelic variant
thereof, or is another variant thereof. In certain embodiments, an INIR12
transgenic locus
comprises or further comprises an insertion and/or substitution of a DNA
element comprising a
cognate guide RNA recognition site (CgRRS) in a junction polynucleotide of
said INIR12
transgenic locus, wherein the CgRRS optionally comprises SEQ ID NO: 37. In
certain
embodiments, transgenic maize plant cells comprising a INIR12 transgenic locus
set forth in SEQ
ID NO: 2, 3, 4, 5, 6, 29, 43, 44, 45, 47, 48, or 49 are provided. Also
provided are transgenic maize
plants and parts thereof including seeds which comprise the aforementioned
transgenic maize plant
cells. INIR12 transgenic maize plants provided herein can exhibit resistance
to fall armyworm
(Spodoptera frugiperda), corn earworm (Helicoverpa zea), western bean cutworm
(Striacosta
albicosta), and black cutworm (Agrotis ipsilon) infestations in comparison to
control maize plants
which lack the Vip3Aa protein.
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[0006] Methods or obtaining a bulked population of inbred seed comprising
selfing any of the
aforementioned INIR12 transgenic maize plants and harvesting seed comprising
the INIR12
transgenic locus from the selfed maize plant are provided.
[0007] Methods of obtaining hybrid maize seed comprising crossing any of the
aforementioned
INIR12 transgenic maize plant to a second maize plant which is genetically
distinct from the first
maize plant and harvesting seed comprising the INIR12 transgenic locus from
the cross are
provided.
[0008] DNA molecules comprising any one of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17,
18, 19, 25, 37, 40, 41, 42, 43, 44, 45, 47, 48, 49, or 50-167 are provided.
Processed transgenic
maize plant products and biological samples comprising the aforementioned DNA
molecules are
also provided. Methods of detecting a maize plant cell comprising a INIR12
transgenic locus
comprising the step of detecting a DNA molecule comprising SEQ ID NO: 7, 8, 9,
10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 25, 37, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, or 50-
167 are also provided.
[0009] Also provided are methods of excising a INIR12 transgenic locus
comprising an CgRRS
and an originator guide RNA recognition site (OgRRS) from the genome of a
maize plant cell
comprising the steps of: (a) contacting a transgenic plant genome of a maize
plant cell comprising
the INIR12 transgenic locus with: (i) an RNA dependent DNA endonuclease
(RdDe); and (ii) a
guide RNA (gRNA) capable of hybridizing to the guide RNA hybridization site of
the OgRRS and
the CgRRS; wherein the RdDe recognizes a OgRRS/gRNA and a CgRRS/gRNA
hybridization
complex; and, (b) selecting a transgenic plant cell, transgenic plant part, or
transgenic plant wherein
the INIR12 transgenic locus flanked by the OgRRS and the CgRRS has been
excised. In certain
embodiments of the methods, the INIR12 locus comprising the CgRRS comprises
the DNSA
sequence set forth in SEQ ID NO: 47 or SEQ ID NO; 48.
[0010] Also provided are methods of making transgenic maize plant cell
comprising an INIR12
transgenic locus comprising: (a) contacting the transgenic plant genome of a
maize MIR162 plant
cell with: (i) a first set of gene editing molecules comprising a first site-
specific nuclease which
introduces a first double stranded DNA break in a 5' junction polynucleotide
of an MIR162
transgenic locus; and (ii) a second set of gene editing molecules comprising a
second site-specific
nuclease which introduces a second double stranded DNA break between the
CaMV35S terminator
element and the ZmUbi promoter of said MIR162 transgenic locus which is
operably linked to
DNA encoding a phosphomannose isomerase (pmi) and a third site specific
nuclease which
introduces a third double stranded DNA break between the DNA encoding the pmi
and DNA
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encoding the nopaline synthase (nos) terminator element of said MIR162
transgenic locus; and (b)
selecting a transgenic maize plant cell, transgenic maize callus, and/or a
transgenic maize plant
comprising an INIR12 transgenic locus wherein one or more nucleotides of said
5' junction
polynucleotide have been deleted and/or substituted, wherein the first
ZmUbiInt promoter, the
vip3Aa19 or vip3Aa20 coding region which is operably linked to the first
ZmUbiInt promoter, the
CaMV 35S terminator element which is operably linked to said vip3Aa19 or
vip3Aa20 coding
region, and the nos terminator element of said MIR162 transgenic locus are
present, and wherein
DNA of said MIR162 transgenic locus comprising a second ZmUbiInt promoter and
an operably
linked phosphomannose isomerase coding region is absent, thereby making a
transgenic maize
plant cell comprising an INIR12 transgenic locus. Transgenic maize plant
cells, transgenic maize
plant callus, transgenic maize plants, and transgenic maize plant seeds
comprising an INIR12
transgenic locus made by the aforementioned methods are also provided. Also
provided are
methods of modifying a transgenic maize plant cell comprising: obtaining a
MIR162 maize event
plant cell, a representative sample of which was deposited at the ATCC under
accession No. PTA-
8166, comprising a nucleotide sequence comprising a first ZmUbiInt promoter, a
vip3Aa19 or
vip3Aa20 coding region which is operably linked to said promoter, a CaMV 35S
terminator
element which is operably linked to said vip3Aa19 or vip3Aa20 coding region, a
second ZmUbiInt
promoter and an operably linked phosphomannose isomerase coding region, and a
nopaline
synthase terminator element; and modifying said nucleotide sequence to
eliminate functionality of
said phosphomannose isomerase coding region and/or to substantially,
essentially, or completely
remove said phosphomannose isomerase coding region, and optionally to
eliminate functionality
of, or substantially, essentially, or completely remove, said second ZmUbiInt
promoter. Also
provided are methods of modifying a transgenic maize plant cell comprising:
obtaining a MIR162
maize event plant cell, a representative sample of which was deposited at the
ATCC under
accession No. PTA-8166, comprising a nucleotide sequence comprising a first
ZmUbiInt promoter,
a vip3Aa19 or vip3Aa20 coding region which is operably linked to said
promoter, a CaMV 35S
terminator element which is operably linked to said vip3Aa19 or vip3Aa20
coding region, a second
ZmUbiInt promoter and an operably linked phosphomannose isomerase coding
region, and a
nopaline synthase terminator element; and modifying said nucleotide sequence
to substantially,
essentially, or completely remove said phosphomannose isomerase coding region,
and optionally
substantially, essentially, or completely remove said second ZmUniInt
promoter.
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BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
100111 Figure 1 shows a diagram of transgene expression cassettes and
selectable markers in the
MIR162 transgenic locus in the deposited seed of ATCC accession No. PTA-8166.
[0012] Figure 2 shows a schematic diagram which compares current breeding
strategies for
introgression of transgenic events (i.e., transgenic loci) to alternative
breeding strategies for
introgression of transgenic events where the transgenic events (i.e.,
transgenic loci) can be removed
following introgression to provide different combinations of transgenic
traits. In Figure 2, "GE"
refers to genome editing (e.g., including introduction of targeted genetic
changes with genome
editing molecules and "Event Removal" refers to excision of a transgenic locus
(i.e., an "Event")
or portion thereof with genome editing molecules.
[0013] Figure 3A, B, C. Figure 3A shows a schematic diagram of a non-limiting
example of: (i)
an untransformed plant chromosome containing non-transgenic DNA which includes
the originator
guide RNA recognition site (OgRRS) (top); (ii) the original transgenic locus
with the OgRRS in
the non-transgenic DNA of the 1st junction polynucleotide (middle); and (iii)
the modified
transgenic locus with a cognate guide RNA inserted into the non-transgenic DNA
of the 2'
junction polynucleotide (bottom). Figure 3B shows a schematic diagram of a non-
limiting example
of a process where a modified transgenic locus with a cognate guide RNA
inserted into the non-
transgenic DNA of the 2nd junction polynucleotide (top) is subjected to
cleavage at the OgRRS and
CgRRS with one guide RNA (gRNA) that hybridizes to gRNA hybridization site in
both the
OgRRS and the CgRRS and an RNA dependent DNA endonuclease (RdDe) that
recognizes and
cleaves the gRNA/OgRRS and the gRNA/CgRRS complex followed by non-homologous
end
joining processes to provide a plant chromosome where the transgenic locus is
excised. Figure 3C
shows a schematic diagram of a non-limiting example of a process where a
modified transgenic
locus with a cognate guide RNA inserted into the non-transgenic DNA of the 2'
junction
polynucleotide (top) is subjected to cleavage at the OgRRS and CgRRS with one
guide RNA
(gRNA) that hybridizes to the gRNA hybridization site in both the OgRRS and
the CgRRS and an
RNA dependent DNA endonuclease (RdDe) that recognizes and cleaves the
gRNA/OgRRS and
the gRNA/CgRRS complex in the presence of a donor DNA template. In Figure 3C,
cleavage of
the modified transgenic locus in the presence of the donor DNA template which
has homology to
non-transgenic DNA but lacks the OgRRS in the 1st and 2nd junction
polynucleotides followed by
homology-directed repair processes to provide a plant chromosome where the
transgenic locus is
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excised and non-transgenic DNA present in the untransformed plant chromosome
is at least
partially restored.
[0014] Figure 4 shows a schematic diagram of the hybridization sites for gRNAs
of SEQ ID NO:
20, 21, and 22. The 5' junction polynucleotide sequence set forth in Figure 4
corresponds to
nucleotides 920 to 1240 of SEQ ID NO: 1 and SEQ ID NO: 46.
[0015] Figure 5A, B, C shows the sequence (SEQ ID NO: 46) of the MIR162 locus
in the deposited
seed of ATCC accession No. PTA-8166 which encodes the Vip3Aa20 protein. The
endogenous
genomic DNA (uppercase), transgenic insert DNA (lowercase) and 5' and 3'
junction sequences
at both ends of the transgenic insert DNA are shown. The OgRRS sequence in the
3' junction
sequence (comprising SEQ ID NO: 27 in the complementary DNA strand) is shown
in bold and
underlined.
[0016] Figure 6 A, B, C shows the sequence (SEQ ID NO: 47) of an INIR12
transgenic locus
obtained by insertion of a CgRRS in the 5' DNA junction polynucleotide
sequence of an MIR 162
transgenic locus in the ATCC accession no. PTA-8166 deposit which encodes the
Vip3Aa20
protein. The endogenous genomic DNA (uppercase) and transgenic insert DNA
(lowercase) as
well as the 5' and 3' junction sequences at both ends of the inserted
transgenic DNA are shown.
The CgRRS sequence comprising the PAM site and gRNA hybridization site in the
5' junction
polynucleotide sequence is shown in bold, lowercase, and underlined. The OgRRS
sequence in the
3' junction polynucleotide sequence (comprising SEQ ID NO: 27 in the
complementary DNA
strand) is shown in bold, uppercase, and underlined.
[0017] Figure 7 A, B shows the sequence (SEQ ID NO: 49) of an INIR12
transgenic locus obtained
by deletion of a 5' DNA junction polynucleotide sequence of an MIR 162
transgenic locus in the
ATCC accession no. PTA-8166 deposit which encodes the Vip3Aa20 protein. The
endogenous
genomic DNA (uppercase) and transgenic insert DNA (lowercase) as well as the
5' and 3' junction
sequences at both ends of the inserted transgenic DNA are shown.
DETAILED DESCRIPTION
[0018] Unless otherwise stated, nucleic acid sequences in the text of this
specification are given,
when read from left to right, in the 5' to 3' direction. Nucleic acid
sequences may be provided as
DNA or as RNA, as specified; disclosure of one necessarily defines the other,
as well as necessarily
defines the exact complements, as is known to one of ordinary skill in the
art.
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[0019] Where a term is provided in the singular, the inventors also
contemplate embodiments
described by the plural of that term.
[0020] The term "about" as used herein means a value or range of values which
would be
understood as an equivalent of a stated value and can be greater or lesser
than the value or range
of values stated by 10 percent. Each value or range of values preceded by the
term "about" is also
intended to encompass the embodiment of the stated absolute value or range of
values.
[0021] The phrase "allelic variant" as used herein refers to a polynucleotide
or polypeptide
sequence variant that occurs in a different strain, variety, or isolate of a
given organism.
[0022] The term "and/or" where used herein is to be taken as specific
disclosure of each of the two
specified features or components with or without the other. Thus, the term
and/or" as used in a
phrase such as "A and/or B" herein is intended to include "A and B," "A or B,"
"A" (alone), and
"B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B,
and/or C" is intended
to encompass each of the following embodiments: A, B, and C; A, B, or C; A or
C; A or B; B or
C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
[0023] As used herein, the phrase "approved transgenic locus" is a genetically
modified plant event
which has been authorized, approved, and/or de-regulated for any one of field
testing, cultivation,
human consumption, animal consumption, and/or import by a governmental body.
Illustrative and
non-limiting examples of governmental bodies which provide such approvals
include the Ministry
of Agriculture of Argentina, Food Standards Australia New Zealand, National
Biosafety Technical
Committee (CTNBio) of Brazil, Canadian Food Inspection Agency, China Ministry
of Agriculture
Biosafety Network, European Food Safety Authority, US Department of
Agriculture, US
Department of Environmental Protection, and US Food and Drug Administration.
[0024] The term "backcross", as used herein, refers to crossing an Fl plant or
plants with one of
the original parents. A backcross is used to maintain or establish the
identity of one parent (species)
and to incorporate a particular trait from a second parent (species). The term
"backcross
generation", as used herein, refers to the offspring of a backcross.
[0025] As used herein, the phrase "biological sample" refers to either intact
or non-intact (e.g.
milled seed or plant tissue, chopped plant tissue, lyophilized tissue) plant
tissue. It may also be an
extract comprising intact or non-intact seed or plant tissue. The biological
sample can comprise
flour, meal, syrup, oil, starch, and cereals manufactured in whole or in part
to contain crop plant
by-products. In certain embodiments, the biological sample is "non-
regenerable" (i.e., incapable
of being regenerated into a plant or plant part). In certain embodiments, the
biological sample refers
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to a homogenate, an extract, or any fraction thereof containing genomic DNA of
the organism from
which the biological sample was obtained, wherein the biological sample does
not comprise living
cells.
[0026] As used herein, the terms "correspond," "corresponding," and the like,
when used in the
context of an nucleotide position, mutation, and/or substitution in any given
polynucleotide (e.g.,
an allelic variant of SEQ ID NO: 1 or SEQ ID NO: 46) with respect to the
reference polynucleotide
sequence (e.g., SEQ ID NO: 1 or SEQ ID NO: 46) all refer to the position of
the polynucleotide
residue in the given sequence that has identity to the residue in the
reference nucleotide sequence
when the given polynucleotide is aligned to the reference polynucleotide
sequence using a pairwise
alignment algorithm (e.g., CLUSTAL 0 1.2.4 with default parameters).
[0027] As used herein, the terms "Cpfl" and "Cas12a" are used interchangeably
to refer to the
same RNA dependent DNA endonuclease (RdDe). Cas12a proteins include the
protein provided
herein as SEQ ID NO: 38.
[0028] The term "crossing" as used herein refers to the fertilization of
female plants (or gametes)
by male plants (or gametes). The term "gamete" refers to the haploid
reproductive cell (egg or
pollen) produced in plants by meiosis from a gametophyte and involved in
sexual reproduction,
during which two gametes of opposite sex fuse to form a diploid zygote. The
term generally
includes reference to a pollen (including the sperm cell) and an ovule
(including the ovum). When
referring to crossing in the context of achieving the introgression of a
genomic region or segment,
the skilled person will understand that in order to achieve the introgression
of only a part of a
chromosome of one plant into the chromosome of another plant, random portions
of the genomes
of both parental lines recombine during the cross due to the occurrence of
crossing-over events in
the production of the gametes in the parent lines. Therefore, the genomes of
both parents must be
combined in a single cell by a cross, where after the production of gametes
from the cell and their
fusion in fertilization will result in an introgression event.
[0029] As used herein, the phrases "DNA junction polynucleotide" and "junction
polynucleotide"
refers to a polynucleotide of about 18 to about 500 base pairs in length
comprised of both
endogenous chromosomal DNA of the plant genome and heterologous transgenic DNA
which is
inserted in the plant genome. A junction polynucleotide can thus comprise
about 8, 10, 20, 50, 100,
200, 250, 500, or 1000 base pairs of endogenous chromosomal DNA of the plant
genome and about
8, 10, 20, 50, 100, 200, 250, 500, or 1000 base pairs of heterologous
transgenic DNA which span
the one end of the transgene insertion site in the plant chromosomal DNA.
Transgene insertion
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sites in chromosomes will typically contain both a 5' junction polynucleotide
and a 3' junction
polynucleotide. In embodiments set forth herein in SEQ ID NO: 1, the 5'
junction polynucleotide
is located at the 5' end of the sequence and the 3' junction polynucleotide is
located at the 3' end
of the sequence. In a non-limiting and illustrative example, a 5' junction
polynucleotide of a
transgenic locus is telomere proximal in a chromosome arm and the 3' junction
polynucleotide of
the transgenic locus is centromere proximal in the same chromosome arm. In
another non-limiting
and illustrative example, a 5' junction polynucleotide of a transgenic locus
is centromere proximal
in a chromosome arm and the 3' junction polynucleotide of the transgenic locus
is telomere
proximal in the same chromosome arm. The junction polynucleotide which is
telomere proximal
and the junction polynucleotide which is centromere proximal can be determined
by comparing
non-transgenic genomic sequence of a sequenced non-transgenic plant genome to
the non-
transgenic DNA in the junction polynucleotides.
[0030] The term "donor," as used herein in the context of a plant, refers to
the plant or plant line
from which the trait, transgenic event, or genomic segment originates, wherein
the donor can have
the trait, introgression, or genomic segment in either a heterozygous or
homozygous state.
[0031] As used herein, the terms "excise" and "delete," when used in the
context of a DNA
molecule, are used interchangeably to refer to the removal of a given DNA
segment or element
(e.g., transgene element or transgenic locus or portion thereof) of the DNA
molecule.
[0032] As used herein, the phrase "elite crop plant" refers to a plant which
has undergone breeding
to provide one or more trait improvements. Elite crop plant lines include
plants which are an
essentially homozygous, e.g. inbred or doubled haploid. Elite crop plants can
include inbred lines
used as is or used as pollen donors or pollen recipients in hybrid seed
production (e.g. used to
produce Fl plants). Elite crop plants can include inbred lines which are
selfed to produce non-
hybrid cultivars or varieties or to produce (e.g., bulk up) pollen donor or
recipient lines for hybrid
seed production. Elite crop plants can include hybrid Fl progeny of a cross
between two distinct
elite inbred or doubled haploid plant lines.
[0033] As used herein, an "event," "a transgenic event," "a transgenic locus"
and related phrases
refer to an insertion of one or more transgenes at a unique site in the genome
of a plant as well as
to DNA fragments, plant cells, plants, and plant parts (e.g., seeds)
comprising genomic DNA
containing the transgene insertion. Such events typically comprise both a 5'
and a 3' DNA junction
polynucleotide and confer one or more useful traits including herbicide
tolerance, insect resistance,
male sterility, and the like.
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[0034] As used herein, the phrases "endogenous sequence," "endogenous gene,"
"endogenous
DNA," "endogenous polynucleotide," and the like refer to the native form of a
polynucleotide,
gene or polypeptide in its natural location in the organism or in the genome
of an organism.
[0035] The terms "exogenous" and "heterologous" as are used synonymously
herein to refer to
any polynucleotide (e.g. DNA molecule) that has been inserted into a new
location in the genome
of a plant. Non-limiting examples of an exogenous or heterologous DNA molecule
include a
synthetic DNA molecule, a non-naturally occurring DNA molecule, a DNA molecule
found in
another species, a DNA molecule found in a different location in the same
species, and/or a DNA
molecule found in the same strain or isolate of a species, where the DNA
molecule has been
inserted into a new location in the genome of a plant.
[0036] As used herein, the term "F1" refers to any offspring of a cross
between two genetically
unlike individuals.
[0037] The term "gene," as used herein, refers to a hereditary unit consisting
of a sequence of DNA
that occupies a specific location on a chromosome and that contains the
genetic instruction for a
particular characteristics or trait in an organism. The term "gene" thus
includes a nucleic acid (for
example, DNA or RNA) sequence that comprises coding sequences necessary for
the production
of an RNA, or a polypeptide or its precursor. A functional polypeptide can be
encoded by a full
length coding sequence or by any portion of the coding sequence as long as the
desired activity or
functional properties (e.g., enzymatic activity, pesticidal activity, ligand
binding, and/or signal
transduction) of the RNA or polypeptide are retained.
[0038] The term "identifying," as used herein with respect to a plant, refers
to a process of
establishing the identity or distinguishing character of a plant, including
exhibiting a certain trait,
containing one or more transgenes, and/or containing one or more molecular
markers.
[0039] As used herein, the term "INIR12" is used herein to refer either
individually or collectively
to items that include any or all of the MIR162 transgenic maize loci which
have been modified as
disclosed herein, transgenic maize plants and parts thereof including seed
that comprise the
modified MIR162 transgenic loci, and DNA obtained therefrom.
[0040] The term "isolated" as used herein means having been removed from its
natural
environment.
[0041] As used herein, the terms "include," "includes," and "including" are to
be construed as at
least having the features to which they refer while not excluding any
additional unspecified
features.
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[0042] As used herein, the phrase "introduced transgene" is a transgene not
present in the original
transgenic locus in the genome of an initial transgenic event or in the genome
of a progeny line
obtained from the initial transgenic event. Examples of introduced transgenes
include exogenous
transgenes which are inserted in a resident original transgenic locus.
[0043] As used herein, the terms "introgression", "introgressed" and
"introgressing" refer to both
a natural and artificial process, and the resulting plants, whereby traits,
genes or DNA sequences
of one species, variety or cultivar are moved into the genome of another
species, variety or cultivar,
by crossing those species. The process may optionally be completed by
backcrossing to the
recurrent parent. Examples of introgression include entry or introduction of a
gene, a transgene, a
regulatory element, a marker, a trait, a trait locus, or a chromosomal segment
from the genome of
one plant into the genome of another plant.
[0044] The phrase "marker-assisted selection", as used herein, refers to the
diagnostic process of
identifying, optionally followed by selecting a plant from a group of plants
using the presence of
a molecular marker as the diagnostic characteristic or selection criterion.
The process usually
involves detecting the presence of a certain nucleic acid sequence or
polymorphism in the genome
of a plant.
[0045] As used herein, the term "MIR162" is used to refer to items that
include a transgenic maize
locus, transgenic maize plants and parts thereof including seed set forth in
US Patent No.
8,455,720, which is incorporated herein by reference in its entirety.
Representative MIR162
transgenic maize seed have been deposited at the American Type Culture
Collection (ATCC,
Manassas, VA, USA) as accession No. PTA-8166. MIR162 transgenic loci include
loci having
the sequence of SEQ ID NO:1, the sequence of the MIR162 locus in the deposited
seed of accession
No. PTA-8166 (SEQ ID NO: 46) and any progeny thereof, as well as allelic
variants and other
variants of SEQ ID NO:1 or SEQ ID NO: 46. Other variants of a MIR162 locus can
include variants
in MIR162 other than those disclosed herein obtained by gene editing
techniques (e.g., by use of
RdDe, CBE, or ABE and gRNAs, TALENs, and/or ZFN).
[0046] The phrase "molecular marker", as used herein, refers to an indicator
that is used in methods
for visualizing differences in characteristics of nucleic acid sequences.
Examples of such indicators
are restriction fragment length polymorphism (RFLP) markers, amplified
fragment length
polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs),
microsatellite markers
(e.g. SSRs), sequence-characterized amplified region (SCAR) markers, Next
Generation
Sequencing (NGS) of a molecular marker, cleaved amplified polymorphic sequence
(CAPS)
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markers or isozyme markers or combinations of the markers described herein
which defines a
specific genetic and chromosomal location.
[0047] As used herein the terms "native" or "natural" define a condition found
in nature. A "native
DNA sequence" is a DNA sequence present in nature that was produced by natural
means or
traditional breeding techniques but not generated by genetic engineering
(e.g., using molecular
biology/transformation techniques).
[0048] The term "offspring", as used herein, refers to any progeny generation
resulting from
crossing, selfing, or other propagation technique.
[0049] The phrase "operably linked" refers to a juxtaposition wherein the
components so described
are in a relationship permitting them to function in their intended manner.
For instance, a promoter
is operably linked to a coding sequence if the promoter affects its
transcription or expression. When
the phrase "operably linked" is used in the context of a PAM site and a guide
RNA hybridization
site, it refers to a PAM site which permits cleavage of at least one strand of
DNA in a
polynucleotide with an RNA dependent DNA endonuclease or RNA dependent DNA
nickase
which recognize the PAM site when a guide RNA complementary to guide RNA
hybridization site
sequences adjacent to the PAM site is present. A OgRRS and its CgRRS, sPAM
sites, or sigRNAR
sites are operably linked to junction polynucleotides when they can be
recognized by a gRNA and
an RdDe to provide for excision of the transgenic locus or portion thereof
flanked by the junction
polynucleotides. When the phrase "operably linked" is used in the context of a
signature PAM site
and a DNA junction polynucleotide, it refers to a PAM site which permits
cleavage of at least one
strand of DNA in the junction polynucleotide with an RNA dependent DNA
endonuclease, RNA
dependent DNA binding protein, or RNA dependent DNA nickase which recognizes
the PAM site
when a guide RNA complementary to sequences adjacent to the PAM site is
present. When the
phrase "operably linked" is used in the context of a sigRNAR site and a DNA
junction
polynucleotide, it refers to a sigRNAR site which permits cleavage of at least
one strand of DNA
in the junction polynucleotide with an RNA dependent DNA endonuclease, RNA
dependent DNA
binding protein, or RNA dependent DNA nickase which recognizes the sigRNAR
site when a guide
RNA complementary to the heterologous sequences adjacent in the sigRNAR site
is present.
[0050] As used herein, the term "plant" includes a whole plant and any
descendant, cell, tissue, or
part of a plant. The term "plant parts" include any part(s) of a plant,
including, for example and
without limitation: seed (including mature seed and immature seed); a plant
cutting; a plant cell; a
plant cell culture; or a plant organ (e.g., pollen, embryos, flowers, fruits,
shoots, leaves, roots,
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stems, and explants). A plant tissue or plant organ may be a seed, protoplast,
callus, or any other
group of plant cells that is organized into a structural or functional unit. A
plant cell or tissue culture
may be capable of regenerating a plant having the physiological and
morphological characteristics
of the plant from which the cell or tissue was obtained, and of regenerating a
plant having
substantially the same genotype as the plant. Regenerable cells in a plant
cell or tissue culture may
be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers,
roots, root tips, silk,
flowers, kernels, ears, cobs, husks, or stalks. In contrast, some plant cells
are not capable of being
regenerated to produce plants and are referred to herein as "non-regenerable"
plant cells.
[0051] The term "purified," as used herein defines an isolation of a molecule
or compound in a
form that is substantially free of contaminants normally associated with the
molecule or compound
in a native or natural environment and means having been increased in purity
as a result of being
separated from other components of the original composition. The term
"purified nucleic acid" is
used herein to describe a nucleic acid sequence which has been separated from
other compounds
including, but not limited to polypeptides, lipids and carbohydrates.
[0052] The term "recipient", as used herein, refers to the plant or plant line
receiving the trait,
transgenic event or genomic segment from a donor, and which recipient may or
may not have the
have trait, transgenic event or genomic segment itself either in a
heterozygous or homozygous
state.
[0053] As used herein the term "recurrent parent" or "recurrent plant"
describes an elite line that
is the recipient plant line in a cross and which will be used as the parent
line for successive
backcrosses to produce the final desired line.
[0054] As used herein the term "recurrent parent percentage" relates to the
percentage that a
backcross progeny plant is identical to the recurrent parent plant used in the
backcross. The percent
identity to the recurrent parent can be determined experimentally by measuring
genetic markers
such as SNPs and/or RFLPs or can be calculated theoretically based on a
mathematical formula.
[0055] The terms "selfed," "selfing," and "self," as used herein, refer to any
process used to obtain
progeny from the same plant or plant line as well as to plants resulting from
the process. As used
herein, the terms thus include any fertilization process wherein both the
ovule and pollen are from
the same plant or plant line and plants resulting therefrom. Typically, the
terms refer to self-
pollination processes and progeny plants resulting from self-pollination.
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[0056] The term "selecting", as used herein, refers to a process of picking
out a certain individual
plant from a group of individuals, usually based on a certain identity, trait,
characteristic, and/or
molecular marker of that individual.
[0057] As used herein, the phrase "originator guide RNA recognition site" or
the acronym
"OgRRS" refers to an endogenous DNA polynucleotide comprising a protospacer
adjacent motif
(PAM) site operably linked to a guide RNA hybridization site. In certain
embodiments, an OgRRS
can be located in an untransformed plant chromosome or in non-transgenic DNA
of a DNA
junction polynucleotide of both an original transgenic locus and a modified
transgenic locus. In
certain embodiments, an OgRRS can be located in transgenic DNA of a DNA
junction
polynucleotide of both an original transgenic locus and a modified transgenic
locus. In certain
embodiments, an OgRRS can be located in both transgenic DNA and non-transgenic
DNA of a
DNA junction polynucleotide of both an original transgenic locus and a
modified transgenic locus
(i.e., can span transgenic and non-transgenic DNA in a DNA junction
polynucleotide).
[0058] As used herein the phrase "cognate guide RNA recognition site" or the
acronym "CgRRS"
refer to a DNA polynucleotide comprising a PAM site operably linked to a guide
RNA
hybridization site, where the CgRRS is absent from transgenic plant genomes
comprising a first
original transgenic locus that is unmodified and where the CgRRS and its
corresponding OgRRS
can hybridize to a single gRNA. A CgRRS can be located in transgenic DNA of a
DNA junction
polynucleotide of a modified transgenic locus, in transgenic DNA of a DNA
junction
polynucleotide of a modified transgenic locus, or in both transgenic and non-
transgenic DNA of a
modified transgenic locus (i.e., can span transgenic and non-transgenic DNA in
a DNA junction
polynucleotide).
[0059] As used herein, the phrase "a transgenic locus excision site" refers to
the DNA which
remains in the genome of a plant or in a DNA molecule (e.g., an isolated or
purified DNA
molecule) wherein a segment comprising, consisting essentially of, or
consisting of a transgenic
locus or portion thereof has been deleted. In a non-limiting and illustrative
example, a transgenic
locus excision site can thus comprise a contiguous segment of DNA comprising
at least 10 base
pairs of DNA that is telomere proximal to the deleted transgenic locus or to
the deleted segment of
the transgenic locus and at least 10 base pairs of DNA that is centromere
proximal to the deleted
transgenic locus or to the deleted segment of the transgenic locus.
[0060] As used herein, the phrase "signature protospacer adjacent motif
(sPAM)" or acronym
"sPAM" refer to a PAM which has been introduced into a transgenic plant genome
by genome
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editing, wherein the sPAM is absent from a transgenic plant genome comprising
the original
transgenic locus. A sPAM can be introduced by an insertion, deletion, and or
substitution of one
or more nucleotides in genomic DNA.
[0061] As used herein the phrase "signature guide RNA Recognition site" or
acronym "sigRNAR
site" refer to a DNA polynucleotide comprising a heterologous crRNA (CRISPR
RNA) binding
sequence located immediately 5' or 3' to a PAM site, wherein the sigRNAR site
has been
introduced into a transgenic plant genome by genome editing and wherein at
least the heterologous
crRNA binding sequence is absent from a transgenic plant genome comprising the
original
transgenic locus. In certain embodiments, the heterologous crRNA binding
sequence is operably
linked to a pre-existing PAM site in the transgenic plant genome. In other
embodiments, the
heterologous crRNA binding sequence is operably linked to a sPAM site in the
transgenic plant
genome.
[0062] As used herein, the phrase "transgene element" refers to a segment of
DNA comprising,
consisting essentially of, or consisting of a promoter, a 5' UTR, an intron, a
coding region, a
3'UTR, or a polyadenylation signal. Polyadenylation signals include transgene
elements referred
to as "terminators" (e.g., NOS, pinII, rbcs, Hsp17, TubA).
[0063] To the extent to which any of the preceding definitions is inconsistent
with definitions
provided in any patent or non-patent reference incorporated herein by
reference, any patent or non-
patent reference cited herein, or in any patent or non-patent reference found
elsewhere, it is
understood that the preceding definition will be used herein.
[0064] Genome editing molecules can permit introduction of targeted genetic
change conferring
desirable traits in a variety of crop plants (Zhang et al. Genome Biol. 2018;
19: 210; Schindele et
al. FEBS Lett. 2018;592(12):1954). Desirable traits introduced into crop
plants such as maize
include herbicide tolerance, improved food and/or feed characteristics, male-
sterility, and drought
stress tolerance. Nonetheless, full realization of the potential of genome
editing methods for crop
improvement will entail efficient incorporation of the targeted genetic
changes in germplasm of
different elite crop plants adapted for distinct growing conditions. Such
elite crop plants will also
desirably comprise useful transgenic loci which confer various traits
including herbicide tolerance,
pest resistance (e.g.; insect, nematode, fungal disease, and bacterial disease
resistance), conditional
male sterility systems for hybrid seed production, abiotic stress tolerance
(e.g., drought tolerance),
improved food and/or feed quality, and improved industrial use (e.g.,
biofuel).
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[0065] INIR12 transgenic loci comprising modifications of a MIR162 transgenic
loci in a maize
plant genome by directed insertion, deletion, and/or substitution of DNA
within or adjacent to such
MIR162 transgenic loci as well as methods of making and using such INIR12
transgenic loci are
provided herein. In certain embodiments, the INIR12 transgenic loci comprise
the first ZmUbiInt
promoter, the vip3Aa19 or vip3Aa20 coding region which is operably linked to
said promoter, the
CaMV 35S terminator element which is operably linked to said vip3Aa19 or
vip3Aa20 coding
region, and the nopaline synthase terminator element of an MIR162 transgenic
locus, wherein
DNA of said MIR162 transgenic locus comprising a second ZmUbiInt promoter and
an operably
linked phosphomannose isomerase (pmi) coding region is absent. Such INIR12
transgenic loci can
thus comprise a vip3Aa19 or vip3Aa20 expression cassette having two tandemly
arrayed
terminator elements (i.e., a CaMV35S and a NOS terminator) while lacking non-
essential DNA
elements (i.e., the duplicate copy of the ZmUbiInt promoter and pmi selectable
marker gene which
is operably linked thereto). Examples of an INIR12 transgenic locus comprising
a vip3Aa20
expression cassette and tandemly arrayed CaMV35S and a NOS terminators include
the INIR12
transgenic loci comprising the DNA sequence of SEQ ID NO: 48 and SEQ ID NO:
49.
[0066] In certain embodiments, INIR12 transgenic loci provided herein can thus
comprise
deletions of selectable marker genes and/or repetitive sequences. In its
unmodified form (in certain
embodiments, the "unmodified form" is the "original form," "original
transgenic locus," etc.) a
MIR162 transgenic locus comprises a phosphomannose isomerase (pmi)-encoding
selectable
marker gene which confers the ability to grow on mannose as a carbon source.
In embodiments
provided herein, the selectable marker gene which is deleted comprises,
consists essentially of, or
consists of a DNA molecule encoding: (i) the phosphomannose isomerase (pmi) of
a MIR162
transgenic locus and the ZmUbi promoter that is operably linked thereto; or
(ii) the
phosphomannose isomerase (pmi) of a MIR162 transgenic locus and both the ZmUbi
promoter
and NOS terminator that are operably linked thereto. In certain embodiments,
DNA elements
comprising the ZmUbi promoter and operably linked pmi coding region
corresponding to at least
nucleotides 5837, 5838, 5840, 5845, or 5850 to 8040, 9060, 9080, 9090, 9100,
or 9105 of SEQ ID
NO:1 or SEQ ID NO:46 can be absent from an INIR12 locus. In certain
embodiments, the INIR12
locus comprising a deletion of DNA encoding the pmi gene and the operably
linked ZmUbi
promoter is set forth in SEQ ID NO: 2, wherein nucleotides designated n in the
sequence are either
absent, independently selected from a guanine, a cytosine, an adenine residue,
or a thymine,
comprise or consist of 1 or more nucleotides corresponding to nucleotides 5831
to 5836 of SEQ
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ID NO:1 or SEQ ID NO:46 and/or comprise or consist of 1 or more nucleotides
corresponding to
nucleotides 9102 to 9107 of SEQ ID NO:1 or SEQ ID NO:46. In certain
embodiments, the deletion
junction sequence present in an INIR12 transgenic locus comprises a DNA
molecule set forth in
SEQ ID NO: 25 which corresponds to nucleotides 5821 to 5850 of SEQ ID NO: 6.
In certain
embodiments, the DNA comprising the ZmUbi promoter and operably linked pmi
coding region
to be deleted is flanked by operably linked protospacer adjacent motif (PAM)
sites in a MIR162
transgenic locus which are recognized by an RNA dependent DNA endonuclease
(RdDe); for
example, a class 2 type II or class 2 type V RdDe. In certain embodiments, the
deleted selectable
marker gene is replaced in an INIR12 transgenic locus by an introduced DNA
sequence as
discussed in further detail elsewhere herein. For example, in certain
embodiments, the introduced
DNA sequence comprises a trait expression cassette such as a trait expression
cassette of another
transgenic locus. In addition to the deletion of a selectable marker gene, in
certain embodiments at
least one copy of a repetitive sequence has also been deleted with genome
editing molecules from
an MIR162 transgenic locus. In certain embodiments, the repetitive sequence
comprises, consists
essentially of, or consists of the two distinct ZmUbiInt promoters which are
each operably linked
to the VIP3Aa20 gene and to the pmi selectable marker gene within the MIR162
transgenic locus
(e.g., as depicted in Figure 1). In certain embodiments, the repetitive
sequence which comprises,
consists essentially of, or consists of the second ZmUbiInt promoter and which
is operably linked
to the pmi selectable maker of an MIR162 transgenic locus is absent from the
INIR12 transgenic
locus. In certain embodiments, any of the aforementioned INIR12 transgenic
loci can optionally
further comprise: (i) an OgRRS and a CgRRS which are operably linked to a 1st
and a 2nd junction
sequence of the INIR12 transgenic locus; (ii) one or more signature
protospacer adjacent motif
(sPAM) sites which are operably linked to a 1st and a 2' junction sequence of
the INIR12
transgenic locus; or (iii) signature guide RNA Recognition site (sigRNAR)
sites which are operably
linked to a 1st and a 2' junction sequence of the INIR12 transgenic locus.
Also provided herein
are plants comprising any of aforementioned INIR12 transgenic loci.
[0067] In certain embodiments, an INIR12 transgenic locus can further comprise
modifications of
a 5' or 3' junction polynucleotide of an MIR162 transgenic locus (e.g., as set
forth in SEQ ID NO:1
or SEQ ID NO:46 and in Figure 1). Such modifications of junction
polynucleotides include
deletions of DNA segments comprising non-essential transgenic DNA in the
junction
polynucleotide. In certain embodiments, such deletions of non-essential DNA of
a 5' junction
polynucleotide of an INIR12 transgenic locus include those set forth in SEQ ID
NO: 3, wherein
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one or more nucleotides in a segment corresponding to nucleotides 1089 to 1098
are absent or
independently selected from A, C, G, or T, with the proviso that the
nucleotides 1089 to 1098 of
SEQ ID NO:3 are not identical to nucleotides 1089 to 1098 of SEQ ID NO:1 or
SEQ ID NO:46.
In certain embodiments, such deletions of non-essential DNA of a 5' junction
polynucleotide of an
INIR12 transgenic locus include those wherein nucleotides corresponding to
nucleotides 1081 to
1104 of SEQ ID NO:3 are: (i) each either absent or independently selected from
a guanine, a
cytosine, an adenine residue, or a thymine residue; (ii) comprise about 2 to 8
consecutive residues
of nucleotides 1,081 to 1092 of SEQ ID NO:1 or SEQ ID NO:46 and/or about 2 to
8 consecutive
residues of nucleotides 1093 to 1104 of SEQ ID NO:1 or SEQ ID NO:46; or (iii)
any combination
of (i) and (ii), wherein each of (i), (ii), and (iii) are with the proviso
that the nucleotides
corresponding to nucleotides 1081 to 1104 of SEQ ID NO: 3 are not identical to
nucleotides 1081
to 1104 of SEQ ID NO:1 or SEQ ID NO:46. In certain embodiments, such deletions
of non-
essential DNA of a 5' junction polynucleotide of an INIR12 transgenic locus
include those wherein
nucleotides corresponding to nucleotides 1081 to 1104 of SEQ ID NO:3 are set
forth in SEQ ID
NO: 7, wherein n is absent, is independently selected from A, C, G, or T,
correspond to 1 to 10
residues of nucleotides 1083 to 1092 of SEQ ID NO:1 or SEQ ID NO:46, and/or
correspond to 1
to 10 residues of nucleotides 1093 to 1102 of SEQ ID NO:1 or SEQ ID NO:46 with
the proviso
that nucleotides corresponding to nucleotide 3 to 22 of SEQ ID NO: 7 are not
identical to residues
1083 to 1102 of SEQ ID NO:1 or SEQ ID NO:46. In certain embodiments, such
deletions of non-
essential DNA of a 5' junction polynucleotide of an INIR12 transgenic locus
include those wherein
nucleotides corresponding to nucleotides 1081 to 1104 of SEQ ID NO:3 are set
forth in SEQ ID
NO: 8, wherein n is absent, is independently selected from A, C, G, or T,
correspond to 1 to 5
residues of nucleotides 1088 to1092 of SEQ ID NO:1 or SEQ ID NO:46, and/or
correspond to 1
to 5 residues of nucleotides 1093 to 1097 of SEQ ID NO:1 or SEQ ID NO:46 with
the proviso that
nucleotides corresponding to residues 8 to 17 of SEQ ID NO: 8 are not
identical to residues 1088
to 1097 of SEQ ID NO:1 or SEQ ID NO:46. In certain embodiments, such deletions
of non-
essential DNA of a 5' junction polynucleotide of an INIR12 transgenic locus
include those wherein
nucleotides corresponding to nucleotides 1081 to 1104 of SEQ ID NO:3 are set
forth in SEQ ID
NO: 9; wherein n is absent, is independently selected from A, C, G, or T,
correspond to 1 to 3
residues of nucleotides 1090-1092 of SEQ ID NO:1 or SEQ ID NO:46, and/or
correspond to 1 to
3 residues of nucleotides 1093 to 1095 of SEQ ID NO:1 or SEQ ID NO:46 with the
proviso that
nucleotides corresponding to residues 1090 to 1095 of SEQ ID NO: 9 are not
identical to
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nucleotides 1090 to 1095 of SEQ ID NO:1 or SEQ ID NO:46. In certain
embodiments, such
deletions of non-essential DNA of a 5' junction polynucleotide of an INIR12
transgenic locus
include those wherein nucleotides corresponding to nucleotides 1081 to 1104 of
SEQ ID NO:3 are
set forth in SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In certain
embodiments, such
deletions of non-essential DNA of a 5' junction polynucleotide of an INIR12
transgenic locus
include those set forth in SEQ ID NO: 39, 40, and SEQ ID NO: 50-167. INIR12
transgenic loci
comprising deletions of the non-essential DNA of a 5' junction polynucleotide
are set forth in SEQ
ID NO: 43 and 49.
[0068] Also provided herein are methods whereby targeted genetic changes are
efficiently
combined with desired subsets of transgenic loci in elite progeny plant lines
(e.g., elite inbreds
used for hybrid seed production or for inbred varietal production). Examples
of such methods
include those illustrated in Figure 2. In certain embodiments, INIR12
transgenic loci provided here
are characterized by polynucleotide sequences that can facilitate as necessary
the removal of the
INIR12 transgenic loci from the genome. Useful applications of such INIR12
transgenic loci and
related methods of making include targeted excision of a INIR12 transgenic
locus or portion
thereof in certain breeding lines to facilitate recovery of germplasm with
subsets of transgenic traits
tailored for specific geographic locations and/or grower preferences. Other
useful applications of
such INIR12 transgenic loci and related methods of making include removal of
transgenic traits
from certain breeding lines when it is desirable to replace the trait in the
breeding line without
disrupting other transgenic loci and/or non-transgenic loci. In certain
embodiments, maize
genomes containing INIR12 transgenic loci or portions thereof which can be
selectively excised
with one or more gRNA molecules and RdDe (RNA dependent DNA endonucleases)
which form
gRNA/target DNA complexes. Such selectively excisable INIR12 transgenic loci
can comprise an
originator guide RNA recognition site (OgRRS) which is identified in non-
transgenic DNA,
transgenic DNA, or a combination thereof in of a first junction polynucleotide
of the transgenic
locus and cognate guide RNA recognition site (CgRRS) which is introduced
(e.g., by genome
editing methods) into a second junction polynucleotide of the transgenic locus
and which can
hybridize to the same gRNA as the OgRRS, thereby permitting excision of the
modified transgenic
locus or portions thereof with a single guide RNA (e.g., as shown in Figures
3A and B). In certain
embodiments, an originator guide RNA recognition site (OgRRS) comprises
endogenous DNA
found in untransformed plants and in endogenous non-transgenic DNA ofjunction
polynucleotides
of transgenic plants containing a modified or unmodified transgenic locus. In
certain embodiments,
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an originator guide RNA recognition site (OgRRS) comprises exogenous
transgenic DNA of
junction polynucleotides of transgenic plants containing a modified or
unmodified transgenic
locus. The OgRRS located in non-transgenic DNA, transgenic DNA, or a
combination thereof in
of a first DNA junction polynucleotide is used to design a related cognate
guide RNA recognition
site (CgRRS) which is introduced (e.g., by genome editing methods) into the
second junction
polynucleotide of the transgenic locus. A CgRRS is thus present in junction
polynucleotides of
modified transgenic loci provided herein and is absent from endogenous DNA
found in
untransformed plants and absent from junction sequences of transgenic plants
containing an
unmodified transgenic locus. A CgRRS is also absent from a combination of non-
transgenic and
transgenic DNA found in junction sequences of transgenic plants containing an
unmodified
transgenic locus. In certain embodiments such as those illustrated in the non-
limiting example of
Figure 3, the OgRRS is located in non-transgenic DNA of a 5' junction
polynucleotide and the
CgRRS is introduced into non-transgenic DNA of a 3' junction polynucleotide.
In other
embodiments, the OgRRS can be located in non-transgenic DNA of a 3' junction
polynucleotide
and the CgRRS is introduced into non-transgenic DNA, transgenic DNA, or a
combination thereof
in a 5' junction polynucleotide. Examples of OgRRS polynucleotide sequences in
or near a 3'
junction polynucleotide in an MIR162 transgenic locus include SEQ ID NO: 26,
27, and 28.
OgRRS polynucleotide sequences located in a first junction polynucleotide can
be introduced into
the second junction polynucleotide using donor DNA templates as illustrated in
Figure 3A and as
elsewhere described herein. A donor DNA template for introducing the SEQ ID
NO: 27 OgRRS
into the 5' junction polynucleotide of an MIR162 locus includes the donor DNA
template of SEQ
ID NO: 32. Integration of the SEQ ID NO: 32 donor DNA template into the 5'
junction
polynucleotide of an MIR162 locus can provide an INIR12 locus comprising the
CgRRS sequence
set forth in SEQ ID NO: 37. Integration of the SEQ ID NO: 32 donor DNA
template into the 5'
junction polynucleotide of an MIR162 locus can provide an INIR12 locus set
forth in SEQ ID NO:
44 or 47, wherein the entire phosphomannose isomerase (pmi)-encoding
selectable marker gene is
retained. An INIR12 transgenic locus of SEQ ID NO: 47 comprising the CgRRS
sequence set forth
in SEQ ID NO: 37 in its 5' junction polynucleotide is shown in Figure 6.
Integration of the SEQ
ID NO: 32 donor DNA template into the 5' junction polynucleotide of an MIR162
locus can
provide an INIR12 locus set forth in SEQ ID NO: 45 (encoding Vip3Aa19) or SEQ
ID NO: 48
(encoding Vip3Aa20), wherein the ZmUbiInt promoter and an operably linked
phosphomannose
isomerase coding region of the pmi- encoding selectable marker gene are
absent.
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[0069] Such selectively excisable INIR12 transgenic loci can also comprise
signature protospacer
adjacent motif (sPAM) sites and/or signature guide RNA recognition (sigRNAR)
sites, wherein
the sPAM and/or sigRNAR sites are operably linked to both DNA junction
polynucleotides of the
INIR12 transgenic locus. Such sigRNAR sites can be recognized by RdDe and
suitable guide
RNAs containing crRNA complementary to heterologous DNA sequences adjacent to
a PAM or
sPAM site to provide for cleavage within or near the two junction
polynucleotides. Such
heterologous sequences which introduced at the sigRNAR site are at least 17 or
18 nucleotides in
length and are complementary to the crRNA of a guide RNA. In certain
embodiments, the
heterologous polynucleotide of the sigRNAR is about 17 or 18 to about 24
nucleotides in length.
Non-limiting features of the heterologous DNA sequences in the sigRNAR
include: (i) absence of
significant homology or sequence identity (e.g., less than 50% sequence
identity across the entire
length of the heterologous sequence) to any other endogenous or transgenic
sequences present in
the transgenic plant genome or in other transgenic genomes of the maize plant
being edited (ii)
absence of significant homology or sequence identity (e.g., less than 50%
sequence identity across
the entire length of the heterologous sequence) of a heterologous sequence of
a first sigRNAR site
to a heterologous sequence of a second or third sigRNAR site; and/or (ii)
optimization of the
heterologoussequence for recognition by the RdDe and guide RNA when used in
conjunction with
a particular PAM sequence. In certain embodiments, the sigRNAR sites which are
created are
recognized by the same class of RdDe (e.g., Class 2 type II or Class 2 type V)
or by the sameRdDe
(e.g., both sPAMs or PAMs of the sigRNAR recognized by the same RdDe (e.g.,
Cas9 or Cas 12
RdDe). In certain embodiments, the same sigRNAR sites can be introduced in
both 5' and 3'
junction polynucleotides to permit excision of the INIR12 transgenic locus by
a single guide RNA
and a single RdDe. In certain embodiments, different sets of distinct sigRNAR
sites can be
introduced in the 5' and 3' junction polynucleotides of different transgenic
loci to permit selective
excision of any single transgenic locus by a single guide RNA and a single
RdDe directed to the
distinct sigRNAR sites that flank the transgenic locus. A sigRNAR site can be
created in the plant
genome by inserting the heterologous sequence adjacent to a pre-existing PAM
sequence using
genome editing molecules. A sigRNAR site can be created in the plant genome by
inserting the
heterologous sequence adjacent to a preexisting PAM sequence using genome
editing molecules.
A sigRNAR site also can be created in the plant genome by inserting both the
heterologous
sequence and an associated PAM or sPAM site in a junction polynucleotide. Such
insertions can
be made in non-transgenic plant genomic DNA of the junction polynucleotide, in
the inserted
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transgenic DNA of the junction polynucleotide, or can span the junction
comprising both non-
transgenic plant genomic DNA and inserted transgenic DNA of the junction
polynucleotide. Such
nucleotide insertions can be effected in the plant genome by using gene
editing molecules (e.g.,
RdDe and guide RNAs, RNA dependent nickases and guide RNAs, Zinc Finger
nucleases or
nickases, or TALE nucleases or nickases) which introduce blunt double stranded
breaks or
staggered double stranded breaks in the DNA junction polynucleotides. In the
case of DNA
insertions, the genome editing molecules can also in certain embodiments
further comprise a donor
DNA template or other DNA template which comprises the heterologous
nucleotides for insertion.
Guide RNAs can be directed to the junction polynucleotides by using a pre-
existing PAM site
located within or adjacent to a junction polynucleotide of the transgenic
locus.
[0070] Also provided herein are allelic variants of any of the INIR12
transgenic loci or DNA
molecules provided herein. In certain embodiments, such allelic variants of
INIR12 transgenic loci
include sequences having at least 85%, 90%, 95%, 98%, or 99% sequence identity
across the entire
length or at least 20, 40, 100, 500, 1,000, 2,000, 4,000, 6,000, 8,000, 9,000,
10,000, or 10,500
nucleotides of SEQ ID NO: 2, 3, 4, 5, 6, 29, 43, 44, 45, 47, 48, or 49. In
certain embodiments, such
allelic variants of INIR12 DNA molecules include sequences having at least
85%, 90%, 95%, 98%,
or 99% sequence identity across the entire length of SEQ ID NO: 2, 3, 4, 5, 6,
10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 29, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, or, 50-167.
[0071] Also provided are unique transgenic locus excision sites created by
excision of INIR12
transgenic loci or selectively excisable INIR12 transgenic loci, DNA molecules
comprising the
INIR12 transgenic loci or unique fragments thereof (i.e., fragments of an
INIR12 locus which are
not found in an MIR162 transgenic locus), INIR12 plants comprising the same,
biological samples
containing the DNA, nucleic acid markers adapted for detecting the DNA
molecules, and related
methods of identifying maize plants comprising unique INIR12 transgenic locus
excision sites and
unique fragments of a INIR12 transgenic locus. DNA molecules comprising unique
fragments of
an INIR12 transgenic locus are diagnostic for the presence of an INIR12
transgenic locus or
fragments thereof in a maize plant, maize cell, maize seed, products obtained
therefrom (e.g., seed
meal or stover), and biological samples. DNA molecules comprising unique
fragments of an
INIR12 transgenic locus include DNA molecules comprising modified 5' junction
polynucleotides.
Unique 5' junction polynucleotides of an INIR12 transgenic locus include: (i)
a DNA molecule
comprising nucleotides corresponding to nucleotides 1080 or 1082 to 1102 or
1104 of SEQ ID
NO:1 or SEQ ID NO:46 with the proviso that the DNA molecules is not identical
to residues 1080
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or 1082 to 1102 or 1104 of SEQ ID NO:1 or SEQ ID NO:46); or (ii) any one of
SEQ ID NO: 7, 8,
or 9, with the proviso that the DNA molecules is not identical to residues
1080 or 1082 to 1102 or
1104 of SEQ ID NO:1 or SEQ ID NO:46; or (iii) or SEQ ID NO: 10, 11, 12, 13,
14, 15, 16, 17, 18,
19, 39, or 40. DNA molecules comprising unique fragments of an INIR12
transgenic locus also
include DNA molecules comprising modified junction polynucleotides containing
CgRRS
sequences comprising insertions of OgRRS sequences (e.g., a CgRRS element
comprising SEQ
ID NO: 37). DNA molecules comprising unique fragments of an INIR12 transgenic
locus also
include DNA molecules comprising deletion junctions corresponding to residues
spanning the
deletion of the phosphomannose isomerase coding region and operably linked
ZmUbiInt promoter
in the INIR12 transgenic locus. Such deletion junctions thus comprise one or
more nucleotides
located between the 35S terminator element and the 5' end of the ZmUbiInt
promoter (e.g.,
nucleotides 5839 to 5858 of SEQ ID NO:1 or SEQ ID NO:46) which are directly
joined to (i.e.,
are contiguous with) nucleotides located between or at the 3' terminus of the
pmi coding region
and the 5' end of the NOS terminator in a MIR162 locus (e.g., nucleotides 9040
to 9105 of SEQ
ID NO:1 or SEQ ID NO:46). Examples of unique INIR12 DNA fragment comprising a
such
deletion include nucleotides 5821 to 5850 of SEQ ID NO: 2, wherein one or more
nucleotides
designated n are absent, independently selected from a guanine, a cytosine, an
adenine residue, or
a thymine residue, comprise or consist of 1 or more nucleotides corresponding
to nucleotides 5831
to 5836 of SEQ ID NO:1 or SEQ ID NO:46 and/or comprise or consist of 1 or more
nucleotides
corresponding to nucleotides 9102 to 9107 of SEQ ID NO:1 or SEQ ID NO:46
junction. Another
example of a unique INIR12 DNA fragment comprising such adeletion junction
include SEQ ID
NO: 25, which corresponds to residues 5821 to 5850 of an INIR12 locus set
forth in SEQ ID NO:
6. Another example of a unique INIR12 DNA fragment comprising such a deletion
junction include
SEQ ID NO: 41 and 42. In certain embodiments, any of the aforementioned unique
fragments of
an INIR12 transgenic locus comprise DNA molecules of at least about 18, 20, or
24 nucleotides to
about 30, 50, 100, or 200 nucleotides in length. Also provided herein are
nucleic acid hybridization
probes and primers (e.g., for SNP analysis) adapted for detection of INIR12
transgenic loci which
can comprise all or part of any of the aforementioned DNA molecules and
optionally a detectable
label. Methods and reagents (e.g., nucleic acid markers including nucleic acid
probes and/or
primers) for detecting plants, edited plant genomes, and biological samples
containing DNA
molecules comprising the transgenic loci excision sites and/or non-essential
DNA deletions are
also provided herein. Detection of the DNA molecules can be achieved by any
combination of
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nucleic acid amplification (e.g., PCR amplification), hybridization,
sequencing, and/or mass-
spectrometry based techniques. Methods set forth for detecting junction
nucleic acids in
unmodified transgenic loci set forth in US 20190136331 and US 9,738,904, both
incorporated
herein by reference in their entireties, can be adapted for use in detection
of the nucleic acids
provided herein. In certain embodiments, such detection is achieved by
amplification and/or
hybridization-based detection methods using a method (e.g., selective
amplification primers)
and/or probe (e.g., capable of selective hybridization or generation of a
specific primer extension
product) which specifically recognizes the target DNA molecule (e.g.,
transgenic locus excision
site) but does not recognize DNA from an unmodified transgenic locus. In
certain embodiments,
the hybridization probes can comprise detectable labels (e.g., fluorescent,
radioactive, epitope, and
chemiluminescent labels). In certain embodiments, a single nucleotide
polymorphism detection
assay can be adapted for detection of the target DNA molecule (e.g.,
transgenic locus excision
site). Detection of any of the aforementioned unique DNA fragments comprising
SEQ ID NO: 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 39, 40, 41, and/or 42 in a biological
sample indicates that the
sample contains material from a INIR12 plant or seed.
[0072] Methods provided herein can be used to excise any transgenic locus
where the first and
second junction sequences comprising the endogenous non-transgenic genomic DNA
and the
heterologous transgenic DNA which are joined at the site of transgene
insertion in the plant genome
are known or have been determined. In certain embodiments provided herein,
transgenic loci can
be removed from crop plant lines to obtain crop plant lines with tailored
combinations of transgenic
loci and optionally targeted genetic changes. Such first and second junction
sequences are readily
identified in new transgenic events by inverse PCR techniques using primers
which are
complementary the inserted transgenic sequences. In certain embodiments, the
first and second
junction sequences of transgenic loci are published. An example of a
transgenic locus which can
be improved and used in the methods provided herein is the maize MIR162
transgenic locus. The
maize MIR162 transgenic locus and its transgenic junction sequences are also
depicted in Figure
1. Maize plants comprising the MIR162 transgenic locus and seed thereof have
been cultivated,
been placed in commerce, and have been described in a variety of publications
by various
governmental bodies. Databases which have compiled descriptions of the MIR162
transgenic locus
include the International Service for the Acquisition of Agri-biotech
Applications (ISAAA)
database (available on the world wide web internet site
"isaaa.org/gmapprovaldatabase/event"),
the GenBit LLC database (available on the world wide web internet site
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"genbitgroup.com/en/gmo/gmodatabase"), and the Biosafety Clearing-House (BCH)
database
(available on the http internet site .-_bch.cbd.int/database/organisms").
[0073] Sequences of the junction polynucleotides as well as the transgenic
insert(s) of an original
MIR162 transgenic locus which can be improved by the methods provided herein
are set forth or
otherwise provided in SEQ ID NO: 1, US 8,455,720, the sequence of the MIR162
locus in the
deposited seed of ATCC accession No. PTA-8166 (SEQ ID NO: 46), and elsewhere
in this
disclosure. In certain embodiments provided herein, the MIR162 transgenic
locus set forth in SEQ
ID NO: 1 or present in the deposited seed of ATCC accession No. PTA-8166 (SEQ
ID NO: 46) is
referred to as an original MIR162 transgenic locus. The MIR162 transgenic
locus set forth in SEQ
ID NO:1 encodes the Vip3Aa19 protein. The MIR162 transgenic locus in the
deposited seed of
ATCC accession No. PTA-8166 (SEQ ID NO: 46) encodes the Vip3Aa20 protein. The
Vip3Aa19
and Vip3Aa20 proteins differ by one amino acid residue. The vip3Aa20 gene in
SEQ ID NO: 46
encodes isoleucine at position 129 of the Vip3Aa20 protein rather than the
methionine residue at
position 129 of the Vip3Aa19 protein encoded by the vip3Aa19 gene of SEQ ID
NO: 1. Allelic or
other variants of the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:46, the
patent references
set forth therein and incorporated herein by reference in their entireties,
and elsewhere in this
disclosure which may be present in certain variant MIR162 transgenic plant
loci (e.g., progeny of
deposited seed of accession No. PTA-8166 which contain allelic variants of SEQ
ID NO:1 or SEQ
ID NO:46 or progeny originating from transgenic plant cells comprising the
original MIR162
transgenic set forth in US 8,455,720 which contain allelic variants of SEQ ID
NO:1 or SEQ ID
NO:46) can also be improved by identifying sequences in the variants that
correspond to the
sequences of SEQ ID NO: 1 or SEQ ID NO: 46 by performing a pairwise alignment
(e.g., using
CLUSTAL 0 1.2.4 with default parameters) and making corresponding changes in
the allelic or
other variant sequences. Such allelic or other variant sequences include
sequences having at least
85%, 90%, 95%, 98%, or 99% sequence identity across the entire length or at
least 20, 40, 100,
500, 1,000, 2,000, 4,000, 8,000, 10,000, or 10,579 nucleotides of SEQ ID NO: 1
or SEQ ID NO:
46. Also provided are plants, plant parts including seeds, genomic DNA, and/or
DNA obtained
from INIR12 plants which comprise one or more modifications (e.g., via
insertion of a CgRRS in
a junction polynucleotide sequence) which provide for selective excision of
the INIR12 transgenic
locus or a portion thereof (e.g., the Vip3A coding region and operably linked
promoter). Such
INIR12 transgenic loci can be treated with gene editing molecules (e.g., RdDe
and gRNA(s)) to
obtain plants wherein a segment comprising, consisting essentially of, or
consisting of the INIR12
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transgenic locus or a portion thereof (e.g., the Vip3A coding region and
operably linked promoter)
is deleted. In certain embodiments, the MIR162 transgenic loci set forth in
SEQ ID NO:1 or SEQ
ID NO:46 and allelic variants thereof are further modified by deletion of a
segment of DNA
comprising, consisting essentially of, or consisting of a selectable marker
gene or portions thereof
(e.g, the pmi coding region and operably linked ZmUbi promoter) and/or non-
essential DNA (e.g.,
T-DNA border sequences or anything other than the ZmUbil::VIP3a::t35S
expression cassette) to
obtain INIR12 transgenic loci. In certain embodiments, the INIR12 transgenic
locus comprises a
deletion of the phosphomannose isomerase (PMI) coding region and operably
linked ZmUbi
promoter which are in a MIR162 transgenic locus. Also provided herein are
methods of detecting
plants, genomic DNA, and/or DNA obtained from plants comprising a INIR12
transgenic locus
which contains one or more of a CgRRS, deletions of selectable marker genes,
deletions of non-
essential DNA, and/or a transgenic locus excision site. A first junction
polynucleotide of a MIR162
transgenic locus can comprise either one of the junction polynucleotides found
at the 5' end or the
3' end of any one of the sequences set forth in SEQ ID NO:1 or SEQ ID NO:46,
allelic variants
thereof, or other variants thereof. An OgRRS can be found within non-
transgenic DNA, transgenic
DNA, or a combination thereof in either one of the junction polynucleotides of
any one of SEQ ID
NO:1 or SEQ ID NO:46, allelic variants thereof, or other variants thereof. A
second junction
polynucleotide of a transgenic locus can comprise either one of the junction
polynucleotides found
at the 5' or 3' end of any one of the sequences set forth in SEQ ID NO:1 or
SEQ ID NO:46, allelic
variants thereof, or other variants thereof. A CgRRS can be introduced within
transgenic, non-
transgenic DNA, or a combination thereof of either one of the junction
polynucleotides of any one
of SEQ ID NO:1 or SEQ ID NO:46, allelic variants thereof, or other variants
thereof to obtain an
INIR12 transgenic locus. In certain embodiments, the OgRRS is found in non-
transgenic DNA or
transgenic DNA of the 5' junction polynucleotide of a transgenic locus of any
one of SEQ ID NO:1
or SEQ ID NO:46, allelic variants thereof, or other variants thereof and the
corresponding CgRRS
is introduced into the transgenic DNA, non-transgenic DNA, or a combination
thereof in the 3'
junction polynucleotide of the MIR162 transgenic locus of SEQ ID NO: 1, SEQ ID
NO: 46, allelic
variants thereof, or other variants thereof to obtain an INIR12 transgenic
locus. In other
embodiments, the OgRRS is found in non-transgenic DNA or transgenic DNA of the
3' junction
polynucleotide of the MIR162 transgenic locus of any one of SEQ ID NO: 1, SEQ
ID NO: 46,
allelic variants thereof, or other variants thereof and the corresponding
CgRRS is introduced into
the transgenic DNA, non-transgenic DNA, or a combination thereof in the 5'
junction
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polynucleotide of the transgenic locus of SEQ ID NO: 1, SEQ ID NO: 46, allelic
variants thereof,
or other variants thereof to obtain an INIR12 transgenic locus. Examples of
INIR12 transgenic loci
comprising a CgRRS insertion in a 5' junction polynucleotide include those set
forth in SEQ ID
NO: 44 (encoding Vip3Aa19), SEQ ID NO: 47 (encoding Vip3Aa20), and SEQ ID NO:
48
(encoding Vip3Aa20).
[0074] In certain embodiments, the CgRRS is comprised in whole or in part of
an exogenous DNA
molecule that is introduced into a DNA junction polynucleotide by genome
editing. In certain
embodiments, the guide RNA hybridization site of the CgRRS is operably linked
to a pre-existing
PAM site in the transgenic DNA or non-transgenic DNA of the transgenic plant
genome. In other
embodiments, the guide RNA hybridization site of the CgRRS is operably linked
to a new PAM
site that is introduced in the DNA junction polynucleotide by genome editing.
A CgRRS can be
located in non-transgenic plant genomic DNA of a DNA junction polynucleotide
of an INIR12
transgenic locus, in transgenic DNA of a DNA junction polynucleotide of an
INIR12 transgenic
locus or can span the junction of the transgenic and non-transgenic DNA of a
DNA junction
polynucleotide of an INIR12 transgenic locus. An OgRRS can likewise be located
in non-
transgenic plant genomic DNA of a DNA junction polynucleotide of an INIR12
transgenic locus,
in transgenic DNA of a DNA junction polynucleotide of an INIR12 transgenic
locus or can span
the junction of the transgenic and non-transgenic DNA of a DNA junction
polynucleotide of an
INIR12 transgenic locus.
[0075] Methods provided herein can be used in a variety of breeding schemes to
obtain elite crop
plants comprising subsets of desired modified transgenic loci comprising an
OgRRS and a CgRRS
operably linked to junction polynucleotide sequences and transgenic loci
excision sites where
undesired transgenic loci or portions thereof have been removed (e.g., by use
of the OgRRS and a
CgRRS). Such methods are useful at least insofar as they allow for production
of distinct useful
donor plant lines each having unique sets of modified transgenic loci and, in
some instances,
targeted genetic changes that are tailored for distinct geographies and/or
product offerings. In an
illustrative and non-limiting example, a different product lines comprising
transgenic loci
conferring only two of three types of herbicide tolerance (e.g.., glyphosate,
glufosinate, and
dicamba) can be obtained from a single donor line comprising three distinct
transgenic loci
conferring resistance to all three herbicides. In certain aspects, plants
comprising the subsets of
undesired transgenic loci and transgenic loci excision sites can further
comprise targeted genetic
changes. Such elite crop plants can be inbred plant lines or can be hybrid
plant lines. In certain
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embodiments, at least two transgenic loci (e.g., transgenic loci including an
INIR12 and another
modified transgenic locus wherein an OgRRS and a CgRRS site is operably linked
to a first and a
second junction sequence and optionally a selectable marker gene and/or non-
essential DNA are
deleted) are introgressed into a desired donor line comprising elite crop
plant germplasm and then
subjected to genome editing molecules to recover plants comprising one of the
two introgressed
transgenic loci as well as a transgenic loci excision site introduced by
excision of the other
transgenic locus or portion thereof by the genome editing molecules. In
certain embodiments, the
genome editing molecules can be used to remove a transgenic locus and
introduce targeted genetic
changes in the crop plant genome. Introgression can be achieved by
backcrossing plants
comprising the transgenic loci to a recurrent parent comprising the desired
elite germplasm and
selecting progeny with the transgenic loci and recurrent parent germplasm.
Such backcrosses can
be repeated and/or supplemented by molecular assisted breeding techniques
using SNP or other
nucleic acid markers to select for recurrent parent germplasm until a desired
recurrent parent
percentage is obtained (e.g., at least about 95%, 96%, 97%, 98%, or 99%
recurrent parent
percentage). A non-limiting, illustrative depiction of a scheme for obtaining
plants with both
subsets of transgenic loci and the targeted genetic changes is shown in the
Figure 2 (bottom
"Alternative" panel), where two or more of the transgenic loci ("Event" in
Figure 2) are provided
in Line A and then moved into elite crop plant germplasm by introgression. In
the non-limiting
Figure 2 illustration, introgression can be achieved by crossing a "Line A"
comprising two or more
of the modified transgenic loci to the elite germplasm and then backcrossing
progeny of the cross
comprising the transgenic loci to the elite germplasm as the recurrent parent)
to obtain a "Universal
Donor" (e.g. Line A+ in Figure 2) comprising two or more of the modified
transgenic loci. This
elite germplasm containing the modified transgenic loci (e.g. "Universal
Donor" of Figure 2) can
then be subjected to genome editing molecules which can excise at least one of
the transgenic loci
("Event Removal" in Figure 2) and introduce other targeted genetic changes
("GE" in Figure 2) in
the genomes of the elite crop plants containing one of the transgenic loci and
a transgenic locus
excision site corresponding to the removal site of one of the transgenic loci.
Such selective excision
of transgenic loci or portion thereof can be effected by contacting the genome
of the plant
comprising two transgenic loci with gene editing molecules (e.g., RdDe and
gRNAs, TALENS,
and/or ZFN) which recognize one transgenic loci but not another transgenic
loci. Genome editing
molecules that provide for selective excision of a first modified transgenic
locus comprising an
OgRRS and a CgRRS include a gRNA that hybridizes to the OgRRS and CgRRS of the
first
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modified transgenic locus and an RdDe that recognizes the gRNA/OgRRS and
gRNA/CgRRS
complexes. Distinct plant lines with different subsets of transgenic loci and
desired targeted genetic
changes are thus recovered (e.g., "Line B-1," "Line B-2," and "Line B-3" in
Figure 2). In certain
embodiments, it is also desirable to bulk up populations of inbred elite crop
plants or their seed
comprising the subset of transgenic loci and a transgenic locus excision site
by selfing. In certain
embodiments, inbred progeny of the selfed maize plants comprising the INIR12
transgenic loci
can be used as a pollen donor or recipient for hybrid seed production. Such
hybrid seed and the
progeny grown therefrom can comprise a subset of desired transgenic loci and a
transgenic loci
excision site.
[0076] Hybrid plant lines comprising elite crop plant germplasm, at least one
transgenic locus and
at least one transgenic locus excision site, and in certain aspects,
additional targeted genetic
changes are also provided herein. Methods for production of such hybrid seed
can comprise
crossing elite crop plant lines where at least one of the pollen donor or
recipient comprises at least
the transgenic locus and a transgenic locus excision site and/or additional
targeted genetic changes.
In certain embodiments, the pollen donor and recipient will comprise germplasm
of distinct
heterotic groups and provide hybrid seed and plants exhibiting heterosis. In
certain embodiments,
the pollen donor and recipient can each comprise a distinct transgenic locus
which confers either a
distinct trait (e.g., herbicide tolerance or insect resistance), a different
type of trait (e.g., tolerance
to distinct herbicides or to distinct insects such as coleopteran or
lepidopteran insects), or a different
mode-of-action for the same trait (e.g., resistance to coleopteran insects by
two distinct modes-of-
action or resistance to lepidopteran insects by two distinct modes-of-action).
In certain
embodiments, the pollen recipient will be rendered male sterile or
conditionally male sterile.
Methods for inducing male sterility or conditional male sterility include
emasculation (e.g.,
detasseling), cytoplasmic male sterility, chemical hybridizing agents or
systems, a transgenes or
transgene systems, and/or mutation(s) in one or more endogenous plant genes.
Descriptions of
various male sterility systems that can be adapted for use with the elite crop
plants provided herein
are described in Wan et al. Molecular Plant; 12, 3, (2019):321-342 as well as
in US 8,618,358; US
20130031674; and US 2003188347.
[0077] In certain embodiments, it will be desirable to use genome editing
molecules to make
modified transgenic loci by introducing a CgRRS into the transgenic loci, to
excise modified
transgenic loci comprising an OgRRS and a CgRRS, and/or to make targeted
genetic changes in
elite crop plant or other germplasm. Techniques for effecting genome editing
in crop plants (e.g.,
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maize,) include use of morphogenic factors such as Wuschel (WUS), Ovule
Development Protein
(ODP), and/or Babyboom (BBM) which can improve the efficiency of recovering
plants with
desired genome edits. In some aspects, the morphogenic factor comprises WUS1,
WUS2, WUS3,
WOX2A, WOX4, WOX5, WOX9, BBM2, BMN2, BMN3, and/or ODP2. In certain
embodiments,
compositions and methods for using WUS, BBM, and/or ODP, as well as other
techniques which
can be adapted for effecting genome edits in elite crop plant and other
germplasm, are set forth in
US 20030082813, US 20080134353, US 20090328252, US 20100100981, US
20110165679, US
20140157453, US 20140173775, and US 20170240911, which are each incorporated
by reference
in their entireties. In certain embodiments, the genome edits can be effected
in regenerable plant
parts (e.g., plant embryos) of elite crop plants by transient provision of
gene editing molecules or
polynucleotides encoding the same and do not necessarily require incorporating
a selectable
marker gene into the plant genome (e.g., US 20160208271 and US 20180273960,
both
incorporated herein by reference in their entireties; Svitashev et al. Nat
Commun. 2016; 7:13274).
[0078] In certain embodiments, edited transgenic plant genomes, transgenic
plant cells, parts, or
plants containing those genomes, and DNA molecules obtained therefrom, can
comprise a desired
subset of transgenic loci and/or comprise at least one transgenic locus
excision site. In certain
embodiments, a segment comprising an INIR12 transgenic locus comprising an
OgRRS in non-
transgenic DNA of a 1" junction polynucleotide sequence and a CgRRS in a 2'
junction
polynucleotide sequence is deleted with a gRNA and RdDe that recognize the
OgRRS and the
CgRRS to produce an INIR12 transgenic locus excision site. In certain
embodiments, a segment
comprising an INIR12 transgenic locus comprising a sPAM and/or a sigRNAR site
in a l5tjunction
polynucleotide sequence and a sPAM and/or a sigRNAR in a 2nd junction
polynucleotide sequence
is deleted with at least one gRNA and RdDe that recognize the sPAM and/or a
sigRNAR to produce
an INIR12 transgenic locus excision site. In certain embodiments, the
transgenic locus excision
site can comprise a contiguous segment of DNA comprising at least 10 base
pairs of DNA that is
telomere proximal to the deleted segment of the transgenic locus and at least
10 base pairs of DNA
that is centromere proximal to the deleted segment of the transgenic locus
wherein the transgenic
DNA (i.e., the heterologous DNA) that has been inserted into the crop plant
genome has been
deleted. In certain embodiments where a segment comprising a transgenic locus
has been deleted,
the transgenic locus excision site can comprise a contiguous segment of DNA
comprising at least
base pairs DNA that is telomere proximal to the deleted segment of the
transgenic locus and at
least 10 base pairs of DNA that is centromere proximal DNA to the deleted
segment of the
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transgenic locus wherein the heterologous transgenic DNA and at least 1, 2, 5,
10, 20, 50, or more
base pairs of endogenous DNA located in a 5' junction sequence and/or in a 3'
junction sequence
of the original transgenic locus that has been deleted. In such embodiments
where DNA comprising
the transgenic locus is deleted, a transgenic locus excision site can comprise
at least 10 base pairs
of DNA that is telomere proximal to the deleted segment of the transgenic
locus and at least 10
base pairs of DNA that is centromere proximal to the deleted segment of the
transgenic locus
wherein all of the transgenic DNA is absent and either all or less than all of
the endogenous DNA
flanking the transgenic DNA sequences are present. In certain embodiments
where a segment
consisting essentially of an original transgenic locus has been deleted, the
transgenic locus excision
site can be a contiguous segment of at least 10 base pairs of DNA that is
telomere proximal to the
deleted segment of the transgenic locus and at least 10 base pairs of DNA that
is centromere
proximal to the deleted segment of the transgenic locus wherein less than all
of the heterologous
transgenic DNA that has been inserted into the crop plant genome is excised.
In certain
aforementioned embodiments where a segment consisting essentially of an
original transgenic
locus has been deleted, the transgenic locus excision site can thus contain at
least 1 base pair of
DNA or 1 to about 2 or 5, 8, 10, 20, or 50 base pairs of DNA comprising the
telomere proximal
and/or centromere proximal heterologous transgenic DNA that has been inserted
into the crop plant
genome. In certain embodiments where a segment consisting of an original
transgenic locus has
been deleted, the transgenic locus excision site can contain a contiguous
segment of DNA
comprising at least 10 base pairs of DNA that is telomere proximal to the
deleted segment of the
transgenic locus and at least 10 base pairs of DNA that is centromere proximal
to the deleted
segment of the transgenic locus wherein the heterologous transgenic DNA that
has been inserted
into the crop plant genome is deleted. In certain embodiments where DNA
consisting of the
transgenic locus is deleted, a transgenic locus excision site can comprise at
least 10 base pairs of
DNA that is telomere proximal to the deleted segment of the transgenic locus
and at least 10 base
pairs of DNA that is centromere proximal to the deleted segment of the
transgenic locus wherein
all of the heterologous transgenic DNA that has been inserted into the crop
plant genome is deleted
and all of the endogenous DNA flanking the heterologous sequences of the
transgenic locus is
present. In any of the aforementioned embodiments or in other embodiments, the
continuous
segment of DNA comprising the transgenic locus excision site can further
comprise an insertion
of 1 to about 2, 5, 10, 20, or more nucleotides between the DNA that is
telomere proximal to the
deleted segment of the transgenic locus and the DNA that is centromere
proximal to the deleted
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segment of the transgenic locus. Such insertions can result either from
endogenous DNA repair
and/or recombination activities at the double stranded breaks introduced at
the excision site and/or
from deliberate insertion of an oligonucleotide. Plants, edited plant genomes,
biological samples,
and DNA molecules (e.g., including isolated or purified DNA molecules)
comprising the INIR12
transgenic loci excision sites are provided herein.
[0079] In other embodiments, a segment comprising a INIR12 transgenic locus
(e.g., a transgenic
locus comprising an OgRRS in non-transgenic DNA of a 1" junction sequence and
a CgRRS in a
2nd junction sequence) can be deleted with a gRNA and RdDe that recognize the
OgRRS and the
CgRRS and replaced with DNA comprising the endogenous non-transgenic plant
genomic DNA
present in the genome prior to transgene insertion. A non-limiting example of
such replacements
can be visualized in Figure 3C, where the donor DNA template can comprise the
endogenous non-
transgenic plant genomic DNA present in the genome prior to transgene
insertion along with
sufficient homology to non-transgenic DNA on each side of the excision site to
permit homology-
directed repair. In certain embodiments, the endogenous non-transgenic plant
genomic DNA
present in the genome prior to transgene insertion can be at least partially
restored. In certain
embodiments, the endogenous non-transgenic plant genomic DNA present in the
genome prior to
transgene insertion can be essentially restored such that no more than about
5, 10, or 20 to about
50, 80, or 100nucleotides are changed relative to the endogenous DNA at the
essentially restored
excision site.
[0080] In certain embodiments, edited transgenic plant genomes and transgenic
plant cells, plant
parts, or plants containing those edited genomes, comprising a modification of
an original
transgenic locus, where the modification comprises an OgRRS and a CgRRS which
are operably
linked to a 1" and a 2' junction sequence, respectively or irrespectively, and
optionally further
comprise a deletion of a segment of the original transgenic locus. In certain
embodiments, the
modification comprises two or more separate deletions and/or there is a
modification in two or
more original transgenic plant loci. In certain embodiments, the deleted
segment comprises,
consists essentially of, or consists of a segment of non-essential DNA in the
transgenic locus.
Illustrative examples of non-essential DNA include but are not limited to
synthetic cloning site
sequences, duplications of transgene sequences; fragments of transgene
sequences, and
Agrobacterium right and/or left border sequences. In certain embodiments, the
non-essential DNA
is a duplication and/or fragment of a promoter sequence and/or is not the
promoter sequence
operably linked in the cassette to drive expression of a transgene. In certain
embodiments, excision
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of the non-essential DNA improves a characteristic, functionality, and/or
expression of a transgene
of the transgenic locus or otherwise confers a recognized improvement in a
transgenic plant
comprising the edited transgenic plant genome. In certain embodiments, the non-
essential DNA
does not comprise DNA encoding a selectable marker gene. In certain
embodiments of an edited
transgenic plant genome, the modification comprises a deletion of the non-
essential DNA and a
deletion of a selectable marker gene. The modification producing the edited
transgenic plant
genome could occur by excising both the non-essential DNA and the selectable
marker gene at the
same time, e.g., in the same modification step, or the modification could
occur step-wise. For
example, an edited transgenic plant genome in which a selectable marker gene
has previously been
removed from the transgenic locus can comprise an original transgenic locus
from which a non-
essential DNA is further excised and vice versa. In certain embodiments, the
modification
comprising deletion of the non-essential DNA and deletion of the selectable
marker gene comprises
excising a single segment of the original transgenic locus that comprises both
the non-essential
DNA and the selectable marker gene. Such modification would result in one
excision site in the
edited transgenic genome corresponding to the deletion of both the non-
essential DNA and the
selectable marker gene. In certain embodiments, the modification comprising
deletion of the non-
essential DNA and deletion of the selectable marker gene comprises excising
two or more
segments of the original transgenic locus to achieve deletion of both the non-
essential DNA and
the selectable marker gene. Such modification would result in at least two
excision sites in the
edited transgenic genome corresponding to the deletion of both the non-
essential DNA and the
selectable marker gene. In certain embodiments of an edited transgenic plant
genome, prior to
excision, the segment to be deleted is flanked by operably linked protospacer
adjacent motif (PAM)
sites in the original or unmodified transgenic locus and/or the segment to be
deleted encompasses
an operably linked PAM site in the original or unmodified transgenic locus. In
certain
embodiments, following excision of the segment, the resulting edited
transgenic plant genome
comprises PAM sites flanking the deletion site in the modified transgenic
locus. In certain
embodiments of an edited transgenic plant genome, the modification comprises a
modification of
a MIR162 transgenic locus.
[0081] In certain embodiments, improvements in a transgenic plant locus are
obtained by
introducing a new cognate guide RNA recognition site (CgRRS) which is operably
linked to a
DNA junction polynucleotide of the transgenic locus in the transgenic plant
genome. Such CgRRS
sites can be recognized by RdDe and a single suitable guide RNA directed to
the CgRRS and the
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originator gRNA Recognition Site (OgRRS) to provide for cleavage within the
junction
polynucleotides which flank an INIR12 transgenic locus. In certain
embodiments, the
CgRRS/gRNA and OgRRS/gRNA hybridization complexes are recognized by the same
class of
RdDe (e.g., Class 2 type II or Class 2 type V) or by the same RdDe (e.g., both
the CgRRS/gRNA
and OgRRS/gRNA hybridization complexes recognized by the same Cas9 or Cas 12
RdDe). Such
CgRRS and OgRRS can be recognized by RdDe and suitable guide RNAs containing
crRNA
sufficiently complementary to the guide RNA hybridization site DNA sequences
adjacent to the
PAM site of the CgRRS and the OgRRS to provide for cleavage within or near the
two junction
polynucleotides. Suitable guide RNAs can be in the form of a single gRNA
comprising a crRNA
or in the form of a crRNA/tracrRNA complex. In the case of the OgRRS site, the
PAM and guide
RNA hybridization site are endogenous DNA polynucleotide molecules found in
the plant genome.
In certain embodiments where the CgRRS is introduced into the plant genome by
genome editing,
gRNA hybridization site polynucleotides introduced at the CgRRS are at least
17 or 18 nucleotides
in length and are complementary to the crRNA of a guide RNA. In certain
embodiments, the gRNA
hybridization site sequence of the OgRRS and/or the CgRRS is about 17 or 18 to
about 24
nucleotides in length. The gRNA hybridization site sequence of the OgRRS and
the gRNA
hybridization site of the CgRRS can be of different lengths or comprise
different sequences so long
as there is sufficient complementarity to permit hybridization by a single
gRNA and recognition
by a RdDe that recognizes and cleaves DNA at the gRNA/OgRRS and gRNA/CgRRS
complex. In
certain embodiments, the guide RNA hybridization site of the CgRRS comprise
about a 17 or 18
to about 24 nucleotide sequence which is identical to the guide RNA
hybridization site of the
OgRRS. In other embodiments, the guide RNA hybridization site of the CgRRS
comprise about a
17 or 18 to about 24 nucleotide sequence which has one, two, three, four, or
five nucleotide
insertions, deletions or substitutions when compared to the guide RNA
hybridization site of the
OgRRS. Certain CgRRS comprising a gRNA hybridization site containing has one,
two, three,
four, or five nucleotide insertions, deletions or substitutions when compared
to the guide RNA
hybridization site of the OgRRS can undergo hybridization with a gRNA which is
complementary
to the OgRRS gRNA hybridization site and be cleaved by certain RdDe. Examples
of mismatches
between gRNAs and guide RNA hybridization sites which allow for RdDe
recognition and
cleavage include mismatches resulting from both nucleotide insertions and
deletions in the DNA
which is hybridized to the gRNA (e.g., Lin et al., doi: 10.1093/nar/gku402).
In certain
embodiments, an operably linked PAM site is co-introduced with the gRNA
hybridization site
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polynucleotide at the CgRRS. In certain embodiments, the gRNA hybridization
site
polynucleotides are introduced at a position adjacent to a resident endogenous
PAM sequence in
the junction polynucleotide sequence to form a CgRRS where the gRNA
hybridization site
polynucleotides are operably linked to the endogenous PAM site. In certain
embodiments, non-
limiting features of the OgRRS, CgRRS, and/or the gRNA hybridization site
polynucleotides
thereof include: (i) absence of significant homology or sequence identity
(e.g., less than 50%
sequence identity across the entire length of the OgRRS, CgRRS, and/or the
gRNA hybridization
site sequence) to any other endogenous or transgenic sequences present in the
transgenic plant
genome or in other transgenic genomes of the maize plant being transformed and
edited; (ii)
absence of significant homology or sequence identity (e.g., less than 50%
sequence identity across
the entire length of the sequence) of a sequence of a first OgRRS and a first
CgRRS to a second
OgRRS and a second CgRRS which are operably linked to junction polynucleotides
of a distinct
transgenic locus; (iii) the presence of some sequence identity (e.g., about
25%, 40%, or 50% to
about 60%, 70%, or 80%) between the OgRRS sequence and endogenous sequences
present at the
site where the CgRRS sequence is introduced; and/or (iv) optimization of the
gRNA hybridization
site polynucleotides for recognition by the RdDe and guide RNA when used in
conjunction with a
particular PAM sequence. In certain embodiments, the first and second OgRRS as
well as the first
and second CgRRS are recognized by the same class of RdDe (e.g., Class 2 type
II or Class 2 type
V) or by the same RdDe (e.g., Cas9 or Cas 12 RdDe). In certain embodiments,
the first OgRRS
site in a first junction polynucleotide and the CgRRS introduced in the second
junction
polynucleotide to permit excision of a first transgenic locus by a first
single guide RNA and a
single RdDe. Such nucleotide insertions or genome edits used to introduce
CgRRS in a transgenic
plant genome can be effected in the plant genome by using gene editing
molecules (e.g., RdDe and
guide RNAs, RNA dependent nickases and guide RNAs, Zinc Finger nucleases or
nickases, or
TALE nucleases or nickases) which introduce blunt double stranded breaks or
staggered double
stranded breaks in the DNA junction polynucleotides. In the case of DNA
insertions, the genome
editing molecules can also in certain embodiments further comprise a donor DNA
template or other
DNA template which comprises the heterologous nucleotides for insertion to
form the CgRRS.
Guide RNAs can be directed to the junction polynucleotides by using a pre-
existing PAM site
located within or adjacent to a junction polynucleotide of the transgenic
locus. Non-limiting
examples of such pre-existing PAM sites present in junction polynucleotides,
which can be used
either in conjunction with an inserted heterologous sequence to form a CgRRS
or which can be
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used to create a double stranded break to insert or create a CgRRS, include
PAM sites recognized
by a Cas12a enzyme. Non-limiting examples where a CgRRS are created in a DNA
sequence are
illustrated in Example 2.
[0082] Transgenic loci comprising OgRRS and CgRRS in a first and a second
junction
polynucleotides can be excised from the genomes of transgenic plants by
contacting the transgenic
loci with RdDe or RNA directed nickases, and a suitable guide RNA directed to
the OgRRS and
CgRRS. A non-limiting example where a modified transgenic locus is excised
from a plant genome
by use of a gRNA and an RdDe that recognizes an OgRRS/gRNA and a CgRRS/gRNA
complex
and introduces dsDNA breaks in both junction polynucleotides and repaired by
NHEJ is depicted
in Figure 3B. In the depicted example set forth in Figure 3B, the OgRRS site
and the CgRRS site
are absent from the plant chromosome comprising the transgene excision site
that results from the
process. In other embodiments provided herein where a modified transgenic
locus is excised from
a plant genome by use of a gRNA and an RdDe that recognizes an OgRRS/gRNA and
a
CgRRS/gRNA complex and repaired by NHEJ or microhomology-mediated end joining
(MMEJ),
the OgRRS and/or other non-transgenic sequences that were originally present
prior to transgene
insertion are at least partially or essentially restored.
[0083] In certain embodiments, edited transgenic plant genomes provided herein
can lack one or
more selectable and/or scoreable markers found in an original event
(transgenic locus). Original
MIR162 transgenic loci (events), including those set forth in SEQ ID NO: 1),
US 8,455,720, the
sequence of the MIR162 locus in the deposited seed of accession No. PTA-8166
(SEQ ID NO: 46)
and progeny thereof, contain a selectable phosphomannose isomerase (pmi)
transgene marker
conferring an ability to grow on mannose. Transgenes encoding a phosphomannose
isomerase
(pmi) can confer the ability to grow on mannose. In certain embodiments
provided herein, the
DNA element comprising, consisting essentially of, or consisting of the ZmUbi
promoter which is
operably linked to a pmi coding region of an MIR162 transgenic locus is absent
from an INIR12
transgenic locus, or scoreable marker transgenes can be excised from an
original transgenic locus
by contacting the transgenic locus with one or more gene editing molecules
which introduce double
stranded breaks in the transgenic locus at the 5' and 3' end of the expression
cassette comprising
the selectable marker transgene (e.g., an RdDe and guide RNAs directed to PAM
sites located at
the 5' and 3' end of the expression cassette comprising the selectable marker
transgenes) and
selecting for plant cells, plant parts, or plants wherein the selectable or
scoreable marker has been
excised. In certain embodiments, the selectable or scoreable marker transgene
can be inactivated.
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Inactivation can be achieved by modifications including insertion, deletion,
and/or substitution of
one or more nucleotides in a promoter element, 5' or 3' untranslated region
(UTRs), intron, coding
region, and/or 3' terminator and/or polyadenylation site of the selectable
marker transgene. Such
modifications can inactivate the selectable or scoreable marker transgene by
eliminating or
reducing promoter activity, introducing a missense mutation, and/or
introducing a pre-mature stop
codon. In certain embodiments, the selectable and/or scoreable marker
transgene can be replaced
by an introduced transgene. In certain embodiments, an original transgenic
locus that was
contacted with gene editing molecules which introduce double stranded breaks
in the transgenic
locus at the 5' and 3' end of the expression cassette comprising the
selectable marker and/or
scoreable transgene can also be contacted with a suitable donor DNA template
comprising an
expression cassette flanked by DNA homologous to remaining DNA in the
transgenic locus located
5' and 3' to the selectable marker excision site. In certain embodiments, a
coding region of the
selectable and/or scoreable marker transgene can be replaced with another
coding region such that
the replacement coding region is operably linked to the promoter and 3'
terminator or
polyadenylation site of the selectable and/or scoreable marker transgene.
[0084] In certain embodiments, edited transgenic plant genomes provided herein
can comprise
additional new introduced transgenes (e.g., expression cassettes) inserted
into the transgenic locus
of a given event. Introduced transgenes inserted at the transgenic locus of an
event subsequent to
the event's original isolation can be obtained by inducing a double stranded
break at a site within
an original transgenic locus (e.g., with genome editing molecules including an
RdDe and suitable
guide RNA(s); a suitable engineered zinc-finger nuclease; a TALEN protein and
the like) and
providing an exogenous transgene in a donor DNA template which can be
integrated at the site of
the double stranded break (e.g. by homology-directed repair (HDR) or by non-
homologous end-
joining (NHEJ)). In certain embodiments, an OgRRS and a CgRRS located in a 1st
junction
polynucleotide and a 2nd junction polynucleotide, respectively, can be used to
delete the transgenic
locus and replace it with one or more new expression cassettes. In certain
embodiments, such
deletions and replacements are effected by introducing dsDNA breaks in both
junction
polynucleotides and providing the new expression cassettes on a donor DNA
template (e.g., in
Figure 3C, the donor DNA template can comprise an expression cassette flanked
by DNA
homologous to non-transgenic DNA located telomere proximal and centromere
proximal to the
excision site). Suitable expression cassettes for insertion include DNA
molecules comprising
promoters which are operably linked to DNA encoding proteins and/or RNA
molecules which
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confer useful traits which are in turn operably linked to polyadenylation
sites or terminator
elements. In certain embodiments, such expression cassettes can also comprise
5' UTRs, 3' UTRs,
and/or introns. Useful traits include biotic stress tolerance (e.g., insect
resistance, nematode
resistance, or disease resistance), abiotic stress tolerance (e.g., heat,
cold, drought, and/or salt
tolerance), herbicide tolerance, and quality traits (e.g., improved fatty acid
compositions, protein
content, starch content, and the like). Suitable expression cassettes for
insertion include expression
cassettes which confer insect resistance, herbicide tolerance, biofuel use, or
male sterility traits
contained in any of the transgenic events set forth in US Patent Application
Public. Nos.
20090038026, 20130031674, 20150361446, 20170088904, 20150267221, 201662346688,
and
20200190533 as well as in US Patent Nos. 6342660, 7323556, 8575434, 6040497,
8759618,
7157281, 6852915, 7705216, 10316330, 8618358, 8450561, 8212113, 9428765,
7897748,
8273959, 8093453,8901378, 9994863, 7928296, and 8466346, each of which are
incorporated
herein by reference in their entireties.
[0085] In certain embodiments, INIR12 plants provided herein, including plants
with one or more
transgenic loci, modified transgenic loci, and/or comprising transgenic loci
excision sites can
further comprise one or more targeted genetic changes introduced by one or
more of gene editing
molecules or systems. Also provided are methods where the targeted genetic
changes are
introduced and one or more transgenic loci are removed from plants either in
series or in parallel
(e.g., as set forth in the non-limiting illustration in Figure 2, bottom
"Alternative" panel, where
"GE" can represent targeted genetic changes induced by gene editing molecules
and "Event
Removal" represents excision of one or more transgenic loci with gene editing
molecules). Such
targeted genetic changes include those conferring traits such as improved
yield, improved food
and/or feed characteristics (e.g., improved oil, starch, protein, or amino
acid quality or quantity),
improved nitrogen use efficiency, improved biofuel use characteristics (e.g.,
improved ethanol
production), male sterility/conditional male sterility systems (e.g., by
targeting endogenous M526,
M545 and MSCA1 genes), herbicide tolerance (e.g., by targeting endogenous ALS,
EPSPS, HPPD,
or other herbicide target genes), delayed flowering, non-flowering, increased
biotic stress
resistance (e.g., resistance to insect, nematode, bacterial, or fungal
damage), increased abiotic
stress resistance (e.g., resistance to drought, cold, heat, metal, or salt),
enhanced lodging resistance,
enhanced growth rate, enhanced biomass, enhanced tillering, enhanced
branching, delayed
flowering time, delayed senescence, increased flower number, improved
architecture for high
density planting, improved photosynthesis, increased root mass, increased cell
number, improved
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seedling vigor, improved seedling size, increased rate of cell division,
improved metabolic
efficiency, and increased meristem size in comparison to a control plant
lacking the targeted
genetic change. Types of targeted genetic changes that can be introduced
include insertions,
deletions, and substitutions of one or more nucleotides in the crop plant
genome. Sites in
endogenous plant genes for the targeted genetic changes include promoter,
coding, and non-coding
regions (e.g., 5' UTRs, introns, splice donor and acceptor sites and 3' UTRs).
In certain
embodiments, the targeted genetic change comprises an insertion of a
regulatory or other DNA
sequence in an endogenous plant gene. Non-limiting examples of regulatory
sequences which can
be inserted into endogenous plant genes with gene editing molecules to effect
targeted genetic
changes which confer useful phenotypes include those set forth in US Patent
Application
Publication 20190352655, which is incorporated herein by reference in its
entirety, such as: (a)
auxin response element (AuxRE) sequence; (b) at least one D1-4 sequence
(Ulmasov et al. (1997)
Plant Cell, 9:1963-1971), (c) at least one DRS sequence (Ulmasov et al. (1997)
Plant Cell, 9:1963-
1971); (d) at least one m5-DRS sequence (Ulmasov et al. (1997) Plant Cell,
9:1963-1971); (e) at
least one P3 sequence; (f) a small RNA recognition site sequence bound by a
corresponding small
RNA (e.g., an siRNA, a microRNA (miRNA), a trans-acting siRNA as described in
U.S. Patent
No. 8,030,473, or a phased sRNA as described in U.S. Patent No. 8,404,928;
both of these cited
patents are incorporated by reference herein); (g) a microRNA (miRNA)
recognition site sequence;
(h) the sequence recognizable by a specific binding agent includes a microRNA
(miRNA)
recognition sequence for an engineered miRNA wherein the specific binding
agent is the
corresponding engineered mature miRNA; (i) a transposon recognition sequence;
(j) a sequence
recognized by an ethylene-responsive element binding-factor-associated
amphiphilic repression
(EAR) motif; (k) a splice site sequence (e.g., a donor site, a branching site,
or an acceptor site; see,
for example, the splice sites and splicing signals set forth in the internet
site
lemur[dot]amu[dot]edu[dot]pl/share/ERISdb/home.html); (1) a recombinase
recognition site
sequence that is recognized by a site-specific recombinase; (m) a sequence
encoding an RNA or
amino acid aptamer or an RNA riboswitch, the specific binding agent is the
corresponding ligand,
and the change in expression is upregulation or downregulation; (n) a hormone
responsive element
recognized by a nuclear receptor or a hormone-binding domain thereof; (o) a
transcription factor
binding sequence; and (p) a polycomb response element (see Xiao et al. (2017)
Nature Genetics,
49:1546-1552, doi: 10.1038/ng.3937). Non limiting examples of target maize
genes that can be
subjected to targeted gene edits to confer useful traits include: (a) ZmIPK1
(herbicide tolerant and
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phytate reduced maize; Shukla etal., Nature. 2009; 459:437-41); (b) ZmGL2
(reduced epicuticular
wax in leaves; Char etal. Plant Biotechnol J. 2015; 13:1002); (c) ZmMTL
(induction of haploid
plants; Kelliher et al. Nature. 2017; 542:105); (d) Wx 1 (high amylopectin
content; US
20190032070; incorporated herein by reference in its entirety); (e) TMS5
(thermosensitive male
sterile; Li et al. J Genet Genomics. 2017; 44:465-8); (f) ALS (herbicide
tolerance; Svitashev et al.;
Plant Physiol. 2015; 169:931-45); and (g) ARGOS8 (drought stress tolerance;
Shi et al., Plant
Biotechnol J. 2017;15:207-16). Non-limiting examples of target genes in crop
plants including
maize which can be subjected to targeted genetic changes which confer useful
phenotypes include
those set forth in US Patent Application Nos. 20190352655, 20200199609,
20200157554, and
20200231982, which are each incorporated herein in their entireties; and Zhang
et al. (Genome
Biol. 2018; 19: 210).
[0086] Gene editing molecules of use in methods provided herein include
molecules capable of
introducing a double-strand break ("DSB") or single-strand break ("SSB") in
double-stranded
DNA, such as in genomic DNA or in a target gene located within the genomic DNA
as well as
accompanying guide RNA or donor DNA template polynucleotides. Examples of such
gene editing
molecules include: (a) a nuclease comprising an RNA-guided nuclease, an RNA-
guided DNA
endonuclease or RNA directed DNA endonuclease (RdDe), a class 1 CRISPR type
nuclease
system, a type II Cas nuclease, a Cas9, a nCas9 nickase, a type V Cas
nuclease, a Cas12a nuclease,
a nCas12a nickase, a Cas12d (CasY), a Cas12e (CasX), a Cas12b (C2c1), a Cas12c
(C2c3), a
Cas12i, a Cas12j, a Cas14, an engineered nuclease, a codon-optimized nuclease,
a zinc-finger
nuclease (ZEN) or nickase, a transcription activator-like effector nuclease
(TAL-effector nuclease
or TALEN) or nickase (TALE-nickase), an Argonaute, and a meganuclease or
engineered
meganuclease; (b) a polynucleotide encoding one or more nucleases capable of
effectuating site-
specific alteration (including introduction of a DSB or SSB) of a target
nucleotide sequence; (c) a
guide RNA (gRNA) for an RNA-guided nuclease, or a DNA encoding a gRNA for an
RNA-guided
nuclease; (d) donor DNA template polynucleotides; and (e) other DNA templates
(dsDNA,
ssDNA, or combinations thereof) suitable for insertion at a break in genomic
DNA (e.g., by non-
homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ).
[0087] CRISPR-type genome editing can be adapted for use in the plant cells
and methods
provided herein in several ways. CRISPR elements, e.g., gene editing molecules
comprising
CRISPR endonucleases and CRISPR guide RNAs including single guide RNAs or
guide RNAs in
combination with tracrRNAs or scoutRNA, or polynucleotides encoding the same,
are useful in
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effectuating genome editing without remnants of the CRISPR elements or
selective genetic
markers occurring in progeny. In certain embodiments, the CRISPR elements are
provided directly
to the eukaryotic cell (e.g., plant cells), systems, methods, and compositions
as isolated molecules,
as isolated or semi-purified products of a cell free synthetic process (e.g.,
in vitro translation), or
as isolated or semi-purified products of in a cell-based synthetic process
(e.g., such as in a bacterial
or other cell lysate). In certain embodiments, genome-inserted CRISPR elements
are useful in
plant lines adapted for use in the methods provide herein. In certain
embodiments, plants or plant
cells used in the systems, methods, and compositions provided herein can
comprise a transgene
that expresses a CRISPR endonuclease (e.g., a Cas9, a Cpfl-type or other
CRISPR endonuclease).
In certain embodiments, one or more CRISPR endonucleases with unique PAM
recognition sites
can be used. Guide RNAs (sgRNAs or crRNAs and a tracrRNA) to form an RNA-
guided
endonuclease/guide RNA complex which can specifically bind sequences in the
gDNA target site
that are adjacent to a protospacer adjacent motif (PAM) sequence. The type of
RNA-guided
endonuclease typically informs the location of suitable PAM sites and design
of crRNAs or
sgRNAs. G-rich PAM sites, e.g., 5'-NGG are typically targeted for design of
crRNAs or sgRNAs
used with Cas9 proteins. Examples of PAM sequences include 5'-NGG
(Streptococcus pyogenes),
5'-NNAGAA (Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus
thermophilus
CRISPR3), 5'-NNGRRT or 5'-NNGRR (Staphylococcus aureus Cas9, SaCas9), and 5'-
NNNGATT (Neisseria meningitidis). T-rich PAM sites (e.g., 5'-TTN or 5'-TTTV,
where "V" is
A, C, or G) are typically targeted for design of crRNAs or sgRNAs used with
Cas12a proteins. In
some instances, Cas12a can also recognize a 5'-CTA PAM motif. Other examples
of potential
Cas12a PAM sequences include TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN, ATTN,
TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN, GCCN,
and CCGN (wherein N is defined as any nucleotide). Cpfl (i.e., Cas12a)
endonuclease and
corresponding guide RNAs and PAM sites are disclosed in US Patent Application
Publication
2016/0208243 Al, which is incorporated herein by reference for its disclosure
of DNA encoding
Cpfl endonucleases and guide RNAs and PAM sites. Introduction of one or more
of a wide variety
of CRISPR guide RNAs that interact with CRISPR endonucleases integrated into a
plant genome
or otherwise provided to a plant is useful for genetic editing for providing
desired phenotypes or
traits, for trait screening, or for gene editing mediated trait introgression
(e.g., for introducing a
trait into a new genotype without backcrossing to a recurrent parent or with
limited backcrossing
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to a recurrent parent). Multiple endonucleases can be provided in expression
cassettes with the
appropriate promoters to allow multiple genome site editing.
[0088] CRISPR technology for editing the genes of eukaryotes is disclosed in
US Patent
Application Publications 2016/0138008A1 and US2015/0344912A1, and in US
Patents 8,697,359,
8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418,
8,871,445,
8,889,356, 8,932,814, 8,795,965, and 8,906,616. Cpfl endonuclease and
corresponding guide
RNAs and PAM sites are disclosed in US Patent Application Publication
2016/0208243 Al. Other
CRISPR nucleases useful for editing genomes include Cas12b and Cas12c (see
Shmakov et al.
(2015) Mol. Cell, 60:385 ¨ 397; Harrington et al. (2020) Molecular Cell
doi:10.1016/j.molce1.2020.06.022) and CasX and CasY (see Burstein et al.
(2016) Nature,
doi:10.1038/nature21059; Harrington et al. (2020) Molecular
Cell
doi :10.1016/j .molce1.2020.06.022), or Cas12j (Pausch et
al, (2020) Science
10.1126/science.abb1400). Plant RNA promoters for expressing CRISPR guide RNA
and plant
codon-optimized CRISPR Cas9 endonuclease are disclosed in International Patent
Application
PCT/U52015/018104 (published as WO 2015/131101 and claiming priority to US
Provisional
Patent Application 61/945,700). Methods of using CRISPR technology for genome
editing in
plants are disclosed in US Patent Application Publications US 2015/0082478A1
and US
2015/0059010A1 and in International Patent Application PCT/U52015/038767 Al
(published as
WO 2016/007347 and claiming priority to US Provisional Patent Application
62/023,246). All of
the patent publications referenced in this paragraph are incorporated herein
by reference in their
entirety. In certain embodiments, an RNA-guided endonuclease that leaves a
blunt end following
cleavage of the target site is used. Blunt-end cutting RNA-guided
endonucleases include Cas9,
Cas12c, and Cas 12h (Yan et al., 2019). In certain embodiments, an RNA-guided
endonuclease
that leaves a staggered single stranded DNA overhanging end following cleavage
of the target site
following cleavage of the target site is used. Staggered-end cutting RNA-
guided endonucleases
include Cas12a, Cas12b, and Cas12e.
[0089] The methods can also use sequence-specific endonucleases or sequence-
specific
endonucleases and guide RNAs that cleave a single DNA strand in a dsDNA target
site. Such
cleavage of a single DNA strand in a dsDNA target site is also referred to
herein and elsewhere as
"nicking" and can be effected by various "nickases" or systems that provide
for nicking. Nickases
that can be used include nCas9 (Cas9 comprising a DlOA amino acid
substitution), nCas12a (e.g.,
Cas12a comprising an R1226A amino acid substitution; Yamano et al., 2016),
Cas12i (Yan et al.
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2019), a zinc finger nickase e.g., as disclosed in Kim et al., 2012), a TALE
nickase (e.g., as
disclosed in Wu et al., 2014), or a combination thereof. In certain
embodiments, systems that
provide for nicking can comprise a Cas nuclease (e.g., Cas9 and/or Cas12a) and
guide RNA
molecules that have at least one base mismatch to DNA sequences in the target
editing site (Fu et
al., 2019). In certain embodiments, genome modifications can be introduced
into the target editing
site by creating single stranded breaks (i.e., "nicks") in genomic locations
separated by no more
than about 10, 20, 30, 40, 50, 60, 80, 100, 150, or 200 base pairs of DNA. In
certain illustrative
and non-limiting embodiments, two nickases (i.e., a CAS nuclease which
introduces a single
stranded DNA break including nCas9, nCas12a, Cas12i, zinc finger nickases,
TALE nickases,
combinations thereof, and the like) or nickase systems can directed to make
cuts to nearby sites
separated by no more than about 10, 20, 30, 40, 50, 60, 80 or 100 base pairs
of DNA. In instances
where an RNA guided nickase and an RNA guide are used, the RNA guides are
adjacent to PAM
sequences that are sufficiently close (i.e., separated by no more than about
10, 20, 30, 40, 50, 60,
80, 100, 150, or 200 base pairs of DNA). For the purposes of gene editing,
CRISPR arrays can be
designed to contain one or multiple guide RNA sequences corresponding to a
desired target DNA
sequence; see, for example, Cong et at. (2013) Science, 339:819-823; Ran et
at. (2013) Nature
Protocols, 8:2281 - 2308. At least 16 or 17 nucleotides of gRNA sequence are
required by Cas9
for DNA cleavage to occur; for Cpfl at least 16 nucleotides of gRNA sequence
are needed to
achieve detectable DNA cleavage and at least 18 nucleotides of gRNA sequence
were reported
necessary for efficient DNA cleavage in vitro; see Zetsche et at. (2015) Cell,
163:759 - 771. In
practice, guide RNA sequences are generally designed to have a length of 17 -
24 nucleotides
(frequently 19, 20, or 21 nucleotides) and exact complementarity (i.e.,
perfect base-pairing) to the
targeted gene or nucleic acid sequence; guide RNAs having less than 100%
complementarity to
the target sequence can be used (e.g., a gRNA with a length of 20 nucleotides
and 1 -4 mismatches
to the target sequence) but can increase the potential for off-target effects.
The design of effective
guide RNAs for use in plant genome editing is disclosed in US Patent
Application Publication
2015/0082478 Al, the entire specification of which is incorporated herein by
reference. More
recently, efficient gene editing has been achieved using a chimeric "single
guide RNA"
("sgRNA"), an engineered (synthetic) single RNA molecule that mimics a
naturally occurring
crRNA-tracrRNA complex and contains both a tracrRNA (for binding the nuclease)
and at least
one crRNA (to guide the nuclease to the sequence targeted for editing); see,
for example, Cong et
at. (2013) Science, 339:819 - 823; Xing et at. (2014) BMC Plant Biol., 14:327 -
340. Chemically
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modified sgRNAs have been demonstrated to be effective in genome editing; see,
for example,
Hendel et at. (2015) Nature Biotechnol., 985 ¨ 991. The design of effective
gRNAs for use in
plant genome editing is disclosed in US Patent Application Publication
2015/0082478 Al, the
entire specification of which is incorporated herein by reference.
[0090] Genomic DNA may also be modified via base editing. Both adenine base
editors (ABE)
which convert A/T base pairs to G/C base pairs in genomic DNA as well as
cytosine base pair
editors (CBE) which effect C to T substitutions can be used in certain
embodiments of the methods
provided herein. In certain embodiments, useful ABE and CBE can comprise
genome site specific
DNA binding elements (e.g., RNA-dependent DNA binding proteins including
catalytically
inactive Cas9 and Cas12 proteins or Cas9 and Cas12 nickases) operably linked
to adenine or
cytidine deaminases and used with guide RNAs which position the protein near
the nucleotide
targeted for substitution. Suitable ABE and CBE disclosed in the literature
(Kim, Nat Plants, 2018
Mar;4(3):148-151) can be adapted for use in the methods set forth herein. In
certain embodiments,
a CBE can comprise a fusion between a catalytically inactive Cas9 (dCas9) RNA
dependent DNA
binding protein fused to a cytidine deaminase which converts cytosine (C) to
uridine (U) and
selected guide RNAs, thereby effecting a C to T substitution; see Komor et at.
(2016) Nature,
533:420 ¨ 424. In other embodiments, C to T substitutions are effected with
Cas9 nickase
[Cas9n(D10A)] fused to an improved cytidine deaminase and optionally a
bacteriophage Mu
dsDNA (double-stranded DNA) end-binding protein Gam; see Komor et at., Sci
Adv. 2017 Aug;
3(8):eaa04774. In other embodiments, adenine base editors (ABEs) comprising an
adenine
deaminase fused to catalytically inactive Cas9 (dCas9) or a Cas9 D 1 OA
nickase can be used to
convert A/T base pairs to G/C base pairs in genomic DNA (Gaudelli et al.,
(2017) Nature
551(7681):464-471.
[0091] In certain embodiments, zinc finger nucleases or zinc finger nickases
can also be used in
the methods provided herein. Zinc-finger nucleases are site-specific
endonucleases comprising two
protein domains: a DNA-binding domain, comprising a plurality of individual
zinc finger repeats
that each recognize between 9 and 18 base pairs, and a DNA-cleavage domain
that comprises a
nuclease domain (typically Fokl). The cleavage domain dimerizes in order to
cleave DNA;
therefore, a pair of ZFNs are required to target non-palindromic target
polynucleotides. In certain
embodiments, zinc finger nuclease and zinc finger nickase design methods which
have been
described (Urnov et at. (2010) Nature Rev. Genet., 11:636 ¨ 646; Mohanta et
al. (2017) Genes vol.
8,12: 399; Ramirez et al. Nucleic Acids Res. (2012); 40(12): 5560-5568; Liu et
al. (2013) Nature
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Communications, 4: 2565) can be adapted for use in the methods set forth
herein. The zinc finger
binding domains of the zinc finger nuclease or nickase provide specificity and
can be engineered
to specifically recognize any desired target DNA sequence. The zinc finger DNA
binding domains
are derived from the DNA-binding domain of a large class of eukaryotic
transcription factors called
zinc finger proteins (ZFPs). The DNA-binding domain of ZFPs typically contains
a tandem array
of at least three zinc "fingers" each recognizing a specific triplet of DNA. A
number of strategies
can be used to design the binding specificity of the zinc finger binding
domain. One approach,
termed "modular assembly", relies on the functional autonomy of individual
zinc fingers with
DNA. In this approach, a given sequence is targeted by identifying zinc
fingers for each component
triplet in the sequence and linking them into a multifinger peptide. Several
alternative strategies
for designing zinc finger DNA binding domains have also been developed. These
methods are
designed to accommodate the ability of zinc fingers to contact neighboring
fingers as well as
nucleotide bases outside their target triplet. Typically, the engineered zinc
finger DNA binding
domain has a novel binding specificity, compared to a naturally-occurring zinc
finger protein.
Engineering methods include, for example, rational design and various types of
selection. Rational
design includes, for example, the use of databases of triplet (or quadruplet)
nucleotide sequences
and individual zinc finger amino acid sequences, in which each triplet or
quadruplet nucleotide
sequence is associated with one or more amino acid sequences of zinc fingers
which bind the
particular triplet or quadruplet sequence. See, e.g., US Patents 6,453,242 and
6,534,261, both
incorporated herein by reference in their entirety. Exemplary selection
methods (e.g., phage display
and yeast two-hybrid systems) can be adapted for use in the methods described
herein. In addition,
enhancement of binding specificity for zinc finger binding domains has been
described in US
Patent 6,794,136, incorporated herein by reference in its entirety. In
addition, individual zinc finger
domains may be linked together using any suitable linker sequences. Examples
of linker sequences
are publicly known, e.g., see US Patents 6,479,626; 6,903,185; and 7,153,949,
incorporated herein
by reference in their entirety. The nucleic acid cleavage domain is non-
specific and is typically a
restriction endonuclease, such as Fokl. This endonuclease must dimerize to
cleave DNA. Thus,
cleavage by Fokl as part of a ZFN requires two adjacent and independent
binding events, which
must occur in both the correct orientation and with appropriate spacing to
permit dimer formation.
The requirement for two DNA binding events enables more specific targeting of
long and
potentially unique recognition sites. Fokl variants with enhanced activities
have been described
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and can be adapted for use in the methods described herein; see, e.g., Guo et
at. (2010) 1 Mol.
Biol., 400:96 - 107.
[0092] Transcription activator like effectors (TALEs) are proteins secreted by
certain
Xanthomonas species to modulate gene expression in host plants and to
facilitate the colonization
by and survival of the bacterium. TALEs act as transcription factors and
modulate expression of
resistance genes in the plants. Recent studies of TALEs have revealed the code
linking the
repetitive region of TALEs with their target DNA-binding sites. TALEs comprise
a highly
conserved and repetitive region consisting of tandem repeats of mostly 33 or
34 amino acid
segments. The repeat monomers differ from each other mainly at amino acid
positions 12 and 13.
A strong correlation between unique pairs of amino acids at positions 12 and
13 and the
corresponding nucleotide in the TALE-binding site has been found. The simple
relationship
between amino acid sequence and DNA recognition of the TALE binding domain
allows for the
design of DNA binding domains of any desired specificity. TALEs can be linked
to a non-specific
DNA cleavage domain to prepare genome editing proteins, referred to as TAL-
effector nucleases
or TALENs. As in the case of ZFNs, a restriction endonuclease, such as Fokl,
can be conveniently
used. Methods for use of TALENs in plants have been described and can be
adapted for use in the
methods described herein, see Mahfouz et al. (2011) Proc. Natl. Acad. Sci.
USA, 108:2623 ¨2628;
Mahfouz (2011) GM Crops, 2:99 ¨ 103; and Mohanta et al. (2017) Genes vol.
8,12: 399). TALE
nickases have also been described and can be adapted for use in methods
described herein (Wu et
al.; Biochem Biophys Res Commun. (2014);446(1):261-6; Luo et al; Scientific
Reports 6,
Article number: 20657 (2016)).
[0093] Embodiments of the donor DNA template molecule having a sequence that
is integrated at
the site of at least one double-strand break (DSB) in a genome include double-
stranded DNA, a
single-stranded DNA, a single-stranded DNA/RNA hybrid, and a double-stranded
DNA/RNA
hybrid. In embodiments, a donor DNA template molecule that is a double-
stranded (e.g., a dsDNA
or dsDNA/RNA hybrid) molecule is provided directly to the plant protoplast or
plant cell in the
form of a double-stranded DNA or a double-stranded DNA/RNA hybrid, or as two
single-stranded
DNA (ssDNA) molecules that are capable of hybridizing to form dsDNA, or as a
single-stranded
DNA molecule and a single-stranded RNA (ssRNA) molecule that are capable of
hybridizing to
form a double-stranded DNA/RNA hybrid; that is to say, the double-stranded
polynucleotide
molecule is not provided indirectly, for example, by expression in the cell of
a dsDNA encoded by
a plasmid or other vector. In various non-limiting embodiments of the method,
the donor DNA
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template molecule that is integrated (or that has a sequence that is
integrated) at the site of at least
one double-strand break (DSB) in a genome is double-stranded and blunt-ended;
in other
embodiments the donor DNA template molecule is double-stranded and has an
overhang or "sticky
end" consisting of unpaired nucleotides (e.g., 1, 2, 3, 4, 5, or 6 unpaired
nucleotides) at one
terminus or both termini. In an embodiment, the DSB in the genome has no
unpaired nucleotides
at the cleavage site, and the donor DNA template molecule that is integrated
(or that has a sequence
that is integrated) at the site of the DSB is a blunt-ended double-stranded
DNA or blunt-ended
double-stranded DNA/RNA hybrid molecule, or alternatively is a single-stranded
DNA or a single-
stranded DNA/RNA hybrid molecule. In another embodiment, the DSB in the genome
has one or
more unpaired nucleotides at one or both sides of the cleavage site, and the
donor DNA template
molecule that is integrated (or that has a sequence that is integrated) at the
site of the DSB is a
double-stranded DNA or double-stranded DNA/RNA hybrid molecule with an
overhang or "sticky
end" consisting of unpaired nucleotides at one or both termini, or
alternatively is a single-stranded
DNA or a single-stranded DNA/RNA hybrid molecule; in embodiments, the donor
DNA template
molecule DSB is a double-stranded DNA or double-stranded DNA/RNA hybrid
molecule that
includes an overhang at one or at both termini, wherein the overhang consists
of the same number
of unpaired nucleotides as the number of unpaired nucleotides created at the
site of a DSB by a
nuclease that cuts in an off-set fashion (e.g., where a Cas12 nuclease effects
an off-set DSB with
5-nucleotide overhangs in the genomic sequence, the donor DNA template
molecule that is to be
integrated (or that has a sequence that is to be integrated) at the site of
the DSB is double-stranded
and has 5 unpaired nucleotides at one or both termini). In certain
embodiments, one or both termini
of the donor DNA template molecule contain no regions of sequence homology
(identity or
complementarity) to genomic regions flanking the DSB; that is to say, one or
both termini of the
donor DNA template molecule contain no regions of sequence that is
sufficiently complementary
to permit hybridization to genomic regions immediately adjacent to the
location of the DSB. In
embodiments, the donor DNA template molecule contains no homology to the locus
of the DSB,
that is to say, the donor DNA template molecule contains no nucleotide
sequence that is sufficiently
complementary to permit hybridization to genomic regions immediately adjacent
to the location of
the DSB. In embodiments, the donor DNA template molecule is at least partially
double-stranded
and includes 2-20 base-pairs, e. g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20
base-pairs; in embodiments, the donor DNA template molecule is double-stranded
and blunt-
ended and consists of 2-20 base-pairs, e.g., 2, 3,4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18,
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19, or 20 base-pairs; in other embodiments, the donor DNA template molecule is
double-stranded
and includes 2-20 base-pairs, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20
base-pairs and in addition has at least one overhang or "sticky end"
consisting of at least one
additional, unpaired nucleotide at one or at both termini. In an embodiment,
the donor DNA
template molecule that is integrated (or that has a sequence that is
integrated) at the site of at least
one double-strand break (DSB) in a genome is a blunt-ended double-stranded DNA
or a blunt-
ended double-stranded DNA/RNA hybrid molecule of about 18 to about 300 base-
pairs, or about
20 to about 200 base-pairs, or about 30 to about 100 base-pairs, and having at
least one
phosphorothioate bond between adjacent nucleotides at a 5' end, 3' end, or
both 5' and 3' ends. In
embodiments, the donor DNA template molecule includes single strands of at
least 11, at least 18,
at least 20, at least 30, at least 40, at least 60, at least 80, at least 100,
at least 120, at least 140, at
least 160, at least 180, at least 200, at least 240, at about 280, or at least
320 nucleotides. In
embodiments, the donor DNA template molecule has a length of at least 2, at
least 3, at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at
least 11 base-pairs if double-
stranded (or nucleotides if single-stranded), or between about 2 to about 320
base-pairs if double-
stranded (or nucleotides if single-stranded), or between about 2 to about 500
base-pairs if double-
stranded (or nucleotides if single-stranded), or between about 5 to about 500
base-pairs if double-
stranded (or nucleotides if single-stranded), or between about 5 to about 300
base-pairs if double-
stranded (or nucleotides if single-stranded), or between about 11 to about 300
base-pairs if double-
stranded (or nucleotides if single-stranded), or about 18 to about 300 base-
pairs if double-stranded
(or nucleotides if single-stranded), or between about 30 to about 100 base-
pairs if double-stranded
(or nucleotides if single-stranded). In embodiments, the donor DNA template
molecule includes
chemically modified nucleotides (see, e.g., the various modifications of
internucleotide linkages,
bases, and sugars described in Verma and Eckstein (1998) Annu. Rev. Biochem.,
67:99-134); in
embodiments, the naturally occurring phosphodiester backbone of the donor DNA
template
molecule is partially or completely modified with phosphorothioate,
phosphorodithioate, or
methylphosphonate internucleotide linkage modifications, or the donor DNA
template molecule
includes modified nucleoside bases or modified sugars, or the donor DNA
template molecule is
labelled with a fluorescent moiety (e.g., fluorescein or rhodamine or a
fluorescent nucleoside
analogue) or other detectable label (e.g., biotin or an isotope). In another
embodiment, the donor
DNA template molecule contains secondary structure that provides stability or
acts as an aptamer.
Other related embodiments include double-stranded DNA/RNA hybrid molecules,
single-stranded
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DNA/RNA hybrid donor molecules, and single-stranded donor DNA template
molecules
(including single-stranded, chemically modified donor DNA template molecules),
which in
analogous procedures are integrated (or have a sequence that is integrated) at
the site of a double-
strand break. Donor DNA templates provided herein include those comprising
CgRRS sequences
flanked by DNA with homology to a donor DNA template (e.g., SEQ ID NO: 32). In
certain
embodiments, integration of the donor DNA templates can be facilitated by use
of a bacteriophage
lambda exonuclease, a bacteriophage lambda beta SSAP protein, and an E. coli
SSB essentially as
set forth in US Patent Application Publication 20200407754, which is
incorporated herein by
reference in its entirety.
[0094] Donor DNA template molecules used in the methods provided herein
include DNA
molecules comprising, from 5' to 3', a first homology arm, a replacement DNA,
and a second
homology arm, wherein the homology arms containing sequences that are
partially or completely
homologous to genomic DNA (gDNA) sequences flanking a target site-specific
endonuclease
cleavage site in the gDNA. In certain embodiments, the replacement DNA can
comprise an
insertion, deletion, or substitution of 1 or more DNA base pairs relative to
the target gDNA. In an
embodiment, the donor DNA template molecule is double-stranded and perfectly
base-paired
through all or most of its length, with the possible exception of any unpaired
nucleotides at either
terminus or both termini. In another embodiment, the donor DNA template
molecule is double-
stranded and includes one or more non-terminal mismatches or non-terminal
unpaired nucleotides
within the otherwise double-stranded duplex. In an embodiment, the donor DNA
template
molecule that is integrated at the site of at least one double-strand break
(DSB) includes between
2-20 nucleotides in one (if single-stranded) or in both strands (if double-
stranded), e. g., 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides on one
or on both strands, each
of which can be base-paired to a nucleotide on the opposite strand (in the
case of a perfectly base-
paired double-stranded polynucleotide molecule). Such donor DNA templates can
be integrated in
genomic DNA containing blunt and/or staggered double stranded DNA breaks by
homology-
directed repair (HDR). In certain embodiments, a donor DNA template homology
arm can be about
20, 50, 100, 200, 400, or 600 to about 800, or 1000 base pairs in length. In
certain embodiments,
a donor DNA template molecule can be delivered to a plant cell) in a circular
(e.g., a plasmid or a
viral vector including a geminivirus vector) or a linear DNA molecule. In
certain embodiments, a
circular or linear DNA molecule that is used can comprise a modified donor DNA
template
molecule comprising, from 5' to 3', a first copy of the target sequence-
specific endonuclease
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cleavage site sequence, the first homology arm, the replacement DNA, the
second homology arm,
and a second copy of the target sequence-specific endonuclease cleavage site
sequence. Without
seeking to be limited by theory, such modified donor DNA template molecules
can be cleaved by
the same sequence-specific endonuclease that is used to cleave the target site
gDNA of the
eukaryotic cell to release a donor DNA template molecule that can participate
in HDR-mediated
genome modification of the target editing site in the plant cell genome. In
certain embodiments,
the donor DNA template can comprise a linear DNA molecule comprising, from 5'
to 3', a cleaved
target sequence-specific endonuclease cleavage site sequence, the first
homology arm, the
replacement DNA, the second homology arm, and a cleaved target sequence-
specific endonuclease
cleavage site sequence. In certain embodiments, the cleaved target sequence-
specific endonuclease
sequence can comprise a blunt DNA end or a blunt DNA end that can optionally
comprise a 5'
phosphate group. In certain embodiments, the cleaved target sequence-specific
endonuclease
sequence comprises a DNA end having a single-stranded 5' or 3' DNA overhang.
Such cleaved
target sequence-specific endonuclease cleavage site sequences can be produced
by either cleaving
an intact target sequence-specific endonuclease cleavage site sequence or by
synthesizing a copy
of the cleaved target sequence-specific endonuclease cleavage site sequence.
Donor DNA
templates can be synthesized either chemically or enzymatically (e.g., in a
polymerase chain
reaction (PCR)). Donor DNA templates provided herein include those comprising
CgRRS
sequences flanked by DNA with homology to a donor DNA template e (e.g., SEQ ID
NO: 32).
[0095] Various treatments are useful in delivery of gene editing molecules
and/or other molecules
to a MIR162 or INIR12 plant cell. In certain embodiments, one or more
treatments is employed
to deliver the gene editing or other molecules (e.g., comprising a
polynucleotide, polypeptide or
combination thereof) into a eukaryotic or plant cell, e.g., through barriers
such as a cell wall, a
plasma membrane, a nuclear envelope, and/or other lipid bilayer. In certain
embodiments, a
polynucleotide-, polypeptide-, or RNP-containing composition comprising the
molecules are
delivered directly, for example by direct contact of the composition with a
plant cell.
Aforementioned compositions can be provided in the form of a liquid, a
solution, a suspension, an
emulsion, a reverse emulsion, a colloid, a dispersion, a gel, liposomes,
micelles, an injectable
material, an aerosol, a solid, a powder, a particulate, a nanoparticle, or a
combination thereof can
be applied directly to a plant, plant part, plant cell, or plant explant
(e.g., through abrasion or
puncture or otherwise disruption of the cell wall or cell membrane, by
spraying or dipping or
soaking or otherwise directly contacting, by microinjection). For example, a
plant cell or plant
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protoplast is soaked in a liquid genome editing molecule-containing
composition, whereby the
agent is delivered to the plant cell. In certain embodiments, the agent-
containing composition is
delivered using negative or positive pressure, for example, using vacuum
infiltration or application
of hydrodynamic or fluid pressure. In certain embodiments, the agent-
containing composition is
introduced into a plant cell or plant protoplast, e.g., by microinjection or
by disruption or
deformation of the cell wall or cell membrane, for example by physical
treatments such as by
application of negative or positive pressure, shear forces, or treatment with
a chemical or physical
delivery agent such as surfactants, liposomes, or nanoparticles; see, e.g.,
delivery of materials to
cells employing microfluidic flow through a cell-deforming constriction as
described in US
Published Patent Application 2014/0287509, incorporated by reference in its
entirety herein. Other
techniques useful for delivering the agent-containing composition to a
eukaryotic cell, plant cell or
plant protoplast include: ultrasound or sonication; vibration, friction, shear
stress, vortexing,
cavitation; centrifugation or application of mechanical force; mechanical cell
wall or cell
membrane deformation or breakage; enzymatic cell wall or cell membrane
breakage or
permeabilization; abrasion or mechanical scarification (e.g., abrasion with
carborundum or other
particulate abrasive or scarification with a file or sandpaper) or chemical
scarification (e.g.,
treatment with an acid or caustic agent); and electroporation. In certain
embodiments, the agent-
containing composition is provided by bacterially mediated (e.g.,
Agrobacterium sp., Rhizobium
sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp.,
Phyllobacterium
sp.) transfection of the plant cell or plant protoplast with a polynucleotide
encoding the genome
editing molecules (e.g., RNA dependent DNA endonuclease, RNA dependent DNA
binding
protein, RNA dependent nickase, ABE, or CBE, and/or guide RNA); see, e.g.,
Broothaerts et at.
(2005) Nature, 433:629 ¨ 633). Any of these techniques or a combination
thereof are alternatively
employed on the plant explant, plant part or tissue or intact plant (or seed)
from which a plant cell
is optionally subsequently obtained or isolated; in certain embodiments, the
agent-containing
composition is delivered in a separate step after the plant cell has been
isolated.
[0096] In some embodiments, one or more polynucleotides or vectors driving
expression of one
or more genome editing molecules or trait-conferring genes (e.g.; herbicide
tolerance, insect
resistance, and/or male sterility) are introduced into a MIR162 or INIR12
plant cell. In certain
embodiments, a polynucleotide vector comprises a regulatory element such as a
promoter operably
linked to one or more polynucleotides encoding genome editing molecules and/or
trait-conferring
genes. In such embodiments, expression of these polynucleotides can be
controlled by selection
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of the appropriate promoter, particularly promoters functional in a eukaryotic
cell (e.g., plant cell);
useful promoters include constitutive, conditional, inducible, and temporally
or spatially specific
promoters (e.g., a tissue specific promoter, a developmentally regulated
promoter, or a cell cycle
regulated promoter). Developmentally regulated promoters that can be used in
plant cells include
Phospholipid Transfer Protein (PLTP), fructose-1,6-bisphosphatase protein,
NAD(P)-binding
Rossmann-Fold protein, adipocyte plasma membrane-associated protein-like
protein, Rieske [2Fe-
2S] iron-sulfur domain protein, chlororespiratory reduction 6 protein, D-
glycerate 3-kinase,
chloroplastic-like protein, chlorophyll a-b binding protein 7, chloroplastic-
like protein, ultraviolet-
B-repressible protein, Soul heme-binding family protein, Photosystem I
reaction center subunit
psi-N protein, and short-chain dehydrogenase/reductase protein that are
disclosed in US Patent
Application Publication No. 20170121722, which is incorporated herein by
reference in its entirety
and specifically with respect to such disclosure. In certain embodiments, the
promoter is operably
linked to nucleotide sequences encoding multiple guide RNAs, wherein the
sequences encoding
guide RNAs are separated by a cleavage site such as a nucleotide sequence
encoding a microRNA
recognition/cleavage site or a self-cleaving ribozyme (see, e.g., Ferre-
D'Amare and Scott (2014)
Cold Spring Harbor Perspectives Biol., 2:a003574). In certain embodiments, the
promoter is an
RNA polymerase III promoter operably linked to a nucleotide sequence encoding
one or more
guide RNAs. In certain embodiments, the RNA polymerase III promoter is a plant
U6
spliceosomal RNA promoter, which can be native to the genome of the plant cell
or from a different
species, e.g., a U6 promoter from maize, tomato, or soybean such as those
disclosed U.S. Patent
Application Publication 2017/0166912, or a homologue thereof; in an example,
such a promoter is
operably linked to DNA sequence encoding a first RNA molecule including a
Cas12a gRNA
followed by an operably linked and suitable 3' element such as a U6 poly-T
terminator. In another
embodiment, the RNA polymerase III promoter is a plant U3, 75L (signal
recognition particle
RNA), U2, or U5 promoter, or chimerics thereof, e.g., as described in U.S.
Patent Application
Publication 20170166912. In certain embodiments, the promoter operably linked
to one or more
polynucleotides is a constitutive promoter that drives gene expression in
eukaryotic cells (e.g.,
plant cells). In certain embodiments, the promoter drives gene expression in
the nucleus or in an
organelle such as a chloroplast or mitochondrion. Examples of constitutive
promoters for use in
plants include a CaMV 35S promoter as disclosed in US Patents 5,858,742 and
5,322,938, a rice
actin promoter as disclosed in US Patent 5,641,876, a maize chloroplast
aldolase promoter as
disclosed in US Patent 7,151,204, and the nopaline synthase (NOS) and octopine
synthase (OCS)
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promoters from Agrobacterium tumefaciens. In certain embodiments, the promoter
operably
linked to one or more polynucleotides encoding elements of a genome-editing
system is a promoter
from figwort mosaic virus (FMV), a RUBISCO promoter, or a pyruvate phosphate
dikinase
(PPDK) promoter, which is active in photosynthetic tissues. Other contemplated
promoters
include cell-specific or tissue-specific or developmentally regulated
promoters, for example, a
promoter that limits the expression of the nucleic acid targeting system to
germline or reproductive
cells (e.g., promoters of genes encoding DNA ligases, recombinases,
replicases, or other genes
specifically expressed in germline or reproductive cells). In certain
embodiments, the genome
alteration is limited only to those cells from which DNA is inherited in
subsequent generations,
which is advantageous where it is desirable that expression of the genome-
editing system be
limited in order to avoid genotoxicity or other unwanted effects. All of the
patent publications
referenced in this paragraph are incorporated herein by reference in their
entirety.
[0097] Expression vectors or polynucleotides provided herein may contain a DNA
segment
near the 3' end of an expression cassette that acts as a signal to terminate
transcription and
directs polyadenylation of the resultant mRNA and may also support promoter
activity. Such
a 3' element is commonly referred to as a "3'-untranslated region" or "3'-UTR"
or a
c`polyadenylation signal." In some cases, plant gene-based 3' elements (or
terminators) consist
of both the 3'-UTR and downstream non-transcribed sequence (Nuccio et al.,
2015). Useful 3'
elements include: Agrobacterium tumefaciens nos 3', tml 3', tmr 3', tms 3',
ocs 3', and tr7 3'
elements disclosed in US Patent No. 6,090,627, incorporated herein by
reference, and 3'
elements from plant genes such as the heat shock protein 17, ubiquitin, and
fructose-1,6-
biphosphatase genes from wheat (Triticum aestivum), and the glutelin, lactate
dehydrogenase,
and beta-tubulin genes from rice (Oryza sativa), disclosed in US Patent
Application Publication
2002/0192813 Al. All of the patent publications referenced in this paragraph
are incorporated
herein by reference in their entireties.
[0098] In certain embodiments, the MIR162 or INIR12 plant cells used herein
can comprise
haploid, diploid, or polyploid plant cells or plant protoplasts, for example,
those obtained from
a haploid, diploid, or polyploid plant, plant part or tissue, or callus. In
certain embodiments,
plant cells in culture (or the regenerated plant, progeny seed, and progeny
plant) are haploid or
can be induced to become haploid; techniques for making and using haploid
plants and plant
cells are known in the art, see, e.g., methods for generating haploids in
Arabidopsis thaliana
by crossing of a wild-type strain to a haploid-inducing strain that expresses
altered forms of
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the centromere-specific hi stone CENH3, as described by Maruthachalam and Chan
in "How to
make haploid Arabidopsis thaliana", protocol available at
www [dot] op enwetware [dot] org/images/d/d3/Hapl oi d Arab i dop si
s_protocol [dot] p df; (Ravi et
at. (2014) Nature Communications, 5:5334, doi: 10.1038/nc0mm56334). Haploids
can also be
obtained in a wide variety of monocot plants (e.g., maize, wheat, rice,
sorghum, barley) by
crossing a plant comprising a mutated CENH3 gene with a wildtype diploid plant
to generate
haploid progeny as disclosed in US Patent No. 9,215,849, which is incorporated
herein by
reference in its entirety. Haploid-inducing maize lines that can be used to
obtain haploid maize
plants and/or cells include Stock 6, MHI (Moldovian Haploid Inducer),
indeterminate
gametophyte (ig) mutation, KEMS, RWK, ZEM, ZMS, KMS, and well as transgenic
haploid
inducer lines disclosed in US Patent No. 9,677,082, which is incorporated
herein by reference
in its entirety. Examples of haploid cells include but are not limited to
plant cells obtained
from haploid plants and plant cells obtained from reproductive tissues, e.g.,
from flowers,
developing flowers or flower buds, ovaries, ovules, megaspores, anthers,
pollen,
megagametophyte, and microspores. In certain embodiments where the plant cell
or plant
protoplast is haploid, the genetic complement can be doubled by chromosome
doubling (e.g.,
by spontaneous chromosomal doubling by meiotic non-reduction, or by using a
chromosome
doubling agent such as colchicine, oryzalin, trifluralin, pronamide, nitrous
oxide gas, anti-
microtubule herbicides, anti-microtubule agents, and mitotic inhibitors) in
the plant cell or
plant protoplast to produce a doubled haploid plant cell or plant protoplast
wherein the
complement of genes or alleles is homozygous; yet other embodiments include
regeneration of
a doubled haploid plant from the doubled haploid plant cell or plant
protoplast. Another
embodiment is related to a hybrid plant having at least one parent plant that
is a doubled haploid
plant provided by this approach. Production of doubled haploid plants provides
homozygosity
in one generation, instead of requiring several generations of self-crossing
to obtain
homozygous plants. The use of doubled haploids is advantageous in any
situation where there
is a desire to establish genetic purity (i.e., homozygosity) in the least
possible time. Doubled
haploid production can be particularly advantageous in slow-growing plants or
for producing
hybrid plants that are offspring of at least one doubled-haploid plant.
[0099] In certain embodiments, the MIR162 or INIR12 plant cells used in the
methods
provided herein can include non-dividing cells. Such non-dividing cells can
include plant cell
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protoplasts, plant cells subjected to one or more of a genetic and/or
pharmaceutically-induced
cell-cycle blockage, and the like.
[00100] In certain embodiments, the MIR162 or INIR12 plant cells in used
in the
methods provided herein can include dividing cells. Dividing cells can include
those cells
found in various plant tissues including leaves, meristems, and embryos. These
tissues include
but are not limited to dividing cells from young maize leaf, meristems and
scutellar tissue from
about 8 or 10 to about 12 or 14 days after pollination (DAP) embryos. The
isolation of maize
embryos has been described in several publications (Brettschneider, Becker,
and Lorz 1997;
Leduc et al. 1996; Frame et al. 2011; K. Wang and Frame 2009). In certain
embodiments, basal
leaf tissues (e.g., leaf tissues located about 0 to 3 cm from the ligule of a
maize plant; Kirienko,
Luo, and Sylvester 2012) are targeted for HDR-mediated gene editing. Methods
for obtaining
regenerable plant structures and regenerating plants from the NHEJ-, MMEJ-, or
HDR-
mediated gene editing of plant cells provided herein can be adapted from
methods disclosed in
US Patent Application Publication No. 20170121722, which is incorporated
herein by
reference in its entirety and specifically with respect to such disclosure. In
certain
embodiments, single plant cells subjected to the HDR-mediated gene editing
will give rise to
single regenerable plant structures. In certain embodiments, the single
regenerable plant cell
structure can form from a single cell on, or within, an explant that has been
subjected to the
NHEJ-, MMEJ-, or HDR-mediated gene editing.
[00101] In some embodiments, methods provided herein can include the
additional step
of growing or regenerating an INIR12 plant from a INIR12 plant cell that had
been subjected
to the gene editing or from a regenerable plant structure obtained from that
INIR12 plant cell.
In certain embodiments, the plant can further comprise an inserted transgene,
a target gene edit,
or genome edit as provided by the methods and compositions disclosed herein.
In certain
embodiments, callus is produced from the plant cell, and plantlets and plants
produced from
such callus. In other embodiments, whole seedlings or plants are grown
directly from the plant
cell without a callus stage. Thus, additional related aspects are directed to
whole seedlings and
plants grown or regenerated from the plant cell or plant protoplast having a
target gene edit or
genome edit, as well as the seeds of such plants. In certain embodiments
wherein the plant cell
or plant protoplast is subjected to genetic modification (for example, genome
editing by means
of, e.g., an RdDe), the grown or regenerated plant exhibits a phenotype
associated with the
genetic modification. In certain embodiments, the grown or regenerated plant
includes in its
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genome two or more genetic or epigenetic modifications that in combination
provide at least
one phenotype of interest. In certain embodiments, a heterogeneous population
of plant cells
having a target gene edit or genome edit, at least some of which include at
least one genetic or
epigenetic modification, is provided by the method; related aspects include a
plant having a
phenotype of interest associated with the genetic or epigenetic modification,
provided by either
regeneration of a plant having the phenotype of interest from a plant cell or
plant protoplast
selected from the heterogeneous population of plant cells having a target gene
or genome edit,
or by selection of a plant having the phenotype of interest from a
heterogeneous population of
plants grown or regenerated from the population of plant cells having a
targeted genetic edit or
genome edit. Examples of phenotypes of interest include herbicide resistance,
improved
tolerance of abiotic stress (e.g., tolerance of temperature extremes, drought,
or salt) or biotic
stress (e.g., resistance to nematode, bacterial, or fungal pathogens),
improved utilization of
nutrients or water, modified lipid, carbohydrate, or protein composition,
improved flavor or
appearance, improved storage characteristics (e.g., resistance to bruising,
browning, or
softening), increased yield, altered morphology (e.g., floral architecture or
color, plant height,
branching, root structure). In an embodiment, a heterogeneous population of
plant cells having
a target gene edit or genome edit (or seedlings or plants grown or regenerated
therefrom) is
exposed to conditions permitting expression of the phenotype of interest;
e.g., selection for
herbicide resistance can include exposing the population of plant cells having
a target gene edit
or genome edit (or seedlings or plants grown or regenerated therefrom) to an
amount of
herbicide or other substance that inhibits growth or is toxic, allowing
identification and
selection of those resistant plant cells (or seedlings or plants) that survive
treatment. Methods
for obtaining regenerable plant structures and regenerating plants from plant
cells or
regenerable plant structures can be adapted from published procedures (Roest
and Gilissen,
Acta Bot. Neerl., 1989, 38(1), 1-23; Bhaskaran and Smith, Crop Sci. 30(6):1328-
1337; Ikeuchi
et al., Development, 2016, 143: 1442-1451). Methods for obtaining regenerable
plant
structures and regenerating plants from plant cells or regenerable plant
structures can also be
adapted from US Patent Application Publication No. 20170121722, which is
incorporated
herein by reference in its entirety and specifically with respect to such
disclosure. Also
provided are heterogeneous or homogeneous populations of such plants or parts
thereof (e.g.,
seeds), succeeding generations or seeds of such plants grown or regenerated
from the plant
cells or plant protoplasts, having a target gene edit or genome edit.
Additional related aspects
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include a hybrid plant provided by crossing a first plant grown or regenerated
from a plant cell
or plant protoplast having a target gene edit or genome edit and having at
least one genetic or
epigenetic modification, with a second plant, wherein the hybrid plant
contains the genetic or
epigenetic modification; also contemplated is seed produced by the hybrid
plant. Also
envisioned as related aspects are progeny seed and progeny plants, including
hybrid seed and
hybrid plants, having the regenerated plant as a parent or ancestor. The plant
cells and
derivative plants and seeds disclosed herein can be used for various purposes
useful to the
consumer or grower. In other embodiments, processed products are made from the
INIR12
plant or its seeds, including: (a) maize seed meal (defatted or non-defatted);
(b) extracted
proteins, oils, sugars, and starches; (c) fermentation products; (d) animal
feed or human food
products (e.g., feed and food comprising maize seed meal (defatted or non-
defatted) and other
ingredients (e.g., other cereal grains, other seed meal, other protein meal,
other oil, other starch,
other sugar, a binder, a preservative, a humectant, a vitamin, and/or mineral;
(e) a
pharmaceutical; (f) raw or processed biomass (e.g., cellulosic and/or
lignocellulosic material);
and (g) various industrial products.
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EMBODIMENTS
[00102]
Various embodiments of the plants, genomes, methods, biological samples, and
other compositions described herein are set forth in the following sets of
numbered
embodiments.
[00103] la.
A transgenic maize plant cell comprising a first ZmUbiInt promoter, a
vip3Aa19 or vip3Aa20 coding region which is operably linked to said promoter,
a CaMV 35S
terminator element which is operably linked to said vip3Aa19 or vip3Aa20
coding region, and
a nopaline synthase terminator element, wherein said cell does not contain a
second ZmUbiInt
promoter and an operably linked phosphomannose isomerase coding region between
said
terminator elements, optionally wherein: (i) the ZmUbiInt promoter, the
vip3Aa19 or
vip3Aa20 coding region which is operably linked to said promoter, the CaMV 35S
terminator
element which is operably linked to said vip3Aa19 or vip3Aa20 coding region
are located in
the maize plant cell genomic location of the MIR162 transgenic locus; (ii)
wherein a selectable
marker or scoreable is absent from said maize plant cell genomic location,
and/or (iii) wherein
the nopaline synthase terminator element is not separated from the CaMV 35S
terminator
element by DNA encoding a selectable marker protein, a scoreable marker
protein, or a protein
conferring a useful trait.
[00104] lb.
A transgenic maize plant cell comprising a nucleotide sequence comprising
a first ZmUbiInt promoter, a vip3Aa19 or vip3Aa20 coding region which is
operably linked to
said promoter, a CaMV 35S terminator element which is operably linked to said
vip3Aa19 or
vip3Aa20 coding region, and a nopaline synthase terminator element, wherein
said nucleotide
sequence does not contain a second ZmUbiInt promoter and an operably linked
phosphomannose isomerase coding region between said terminator elements
optionally
wherein: (i) the ZmUbiInt promoter, the vip3Aa19 or vip3Aa20 coding region
which is
operably linked to said promoter, the CaMV 35S terminator element which is
operably linked
to said vip3Aa19 or vip3Aa20 coding region are located in the maize plant cell
genomic
location of the MIR162 transgenic locus; (ii) wherein a selectable marker or
scoreable is absent
from said maize plant cell genomic location, and/or (iii) wherein the nopaline
synthase
terminator element is not separated from the CaMV 35S terminator element by
DNA encoding
a selectable marker protein, a scoreable marker protein, or a protein
conferring a useful trait.
[00105] lc.
A transgenic maize plant cell comprising a nucleotide sequence comprising
a ZmUbiInt promoter, a vip3Aa19 or vip3Aa20 coding region which is operably
linked to said
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promoter, a CaMV 35S terminator element which is operably linked to said
vip3Aa19 or
vip3Aa20 coding region, and a nopaline synthase terminator element, wherein
said nucleotide
sequence does not contain a phosphomannose isomerase coding region between
said terminator
elements, optionally wherein: (i) the ZmUbiInt promoter, the vip3Aa19 or
vip3Aa20 coding
region which is operably linked to said promoter, the CaMV 35S terminator
element which is
operably linked to said vip3Aa19 or vip3Aa20 coding region are located in the
maize plant cell
genomic location of the MIR162 transgenic locus; (ii) wherein a selectable
marker or scoreable
is absent from said maize plant cell genomic location, and/or (iii) wherein
the nopaline synthase
terminator element is not separated from the CaMV 35S terminator element by
DNA encoding
a selectable marker protein, a scoreable marker protein, or a protein
conferring a useful trait.
[00106] id. A transgenic maize plant cell comprising a nucleotide sequence
comprising
a first ZmUbiInt promoter, a vip3Aa19 or vip3Aa20 coding region which is
operably linked to
said promoter, a CaMV 35S terminator element which is operably linked to said
vip3Aa19 or
vip3Aa20 coding region, and a nopaline synthase terminator element, wherein
said nucleotide
sequence does not contain a second ZmUbiInt promoter and an operably linked
phosphomannose isomerase coding region, optionally wherein: (i) the ZmUbiInt
promoter, the
vip3Aa19 or vip3Aa20 coding region which is operably linked to said promoter,
the CaMV
35S terminator element which is operably linked to said vip3Aa19 or vip3Aa20
coding region
are located in the maize plant cell genomic location of the MIR162 transgenic
locus; (ii)
wherein a selectable marker or scoreable is absent from said maize plant cell
genomic location,
and/or (iii) wherein the nopaline synthase terminator element is not separated
from the CaMV
35S terminator element by DNA encoding a selectable marker protein, a
scoreable marker
protein, or a protein conferring a useful trait.
[00107] le. A transgenic maize plant cell comprising a nucleotide sequence
comprising
a ZmUbiInt promoter, a vip3Aa19 or vip3Aa20 coding region which is operably
linked to said
promoter, a CaMV 35S terminator element which is operably linked to said
vip3Aa19 or
vip3Aa20 coding region, and a nopaline synthase terminator element, wherein
said nucleotide
sequence does not contain a phosphomannose isomerase coding region, optionally
wherein: (i)
the ZmUbiInt promoter, the vip3Aa19 or vip3Aa20 coding region which is
operably linked to
said promoter, the CaMV 35S terminator element which is operably linked to
said vip3Aa19
or vip3Aa20 coding region are located in the maize plant cell genomic location
of the MIR162
transgenic locus; (ii) wherein a selectable marker or scoreable is absent from
said maize plant
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cell genomic location, and/or (iii) wherein the nopaline synthase terminator
element is not
separated from the CaMV 35S terminator element by DNA encoding a selectable
marker
protein, a scoreable marker protein, or a protein conferring a useful trait.
[00108] if. A transgenic maize plant cell comprising an INIR12 transgenic
locus
comprising the first ZmUbiInt promoter, the vip3Aa19 or vip3Aa20 coding region
which is
operably linked to said promoter, the CaMV 35S terminator element which is
operably linked
to said vip3Aa19 or vip3Aa20 coding region, and the nopaline synthase
terminator element of
a MIR162 transgenic locus, allelic variants thereof, or other variants
thereof, wherein DNA of
said original MIR162 transgenic locus, allelic variants thereof, or other
variants thereof
comprising a second ZmUbiInt promoter and an operably linked phosphomannose
isomerase
coding region is absent.
[00109] lg. A transgenic maize plant cell comprising an INIR12 transgenic
locus
comprising an insertion and/or substitution of a DNA element comprising a
cognate guide RNA
recognition site (CgRRS) in a DNA junction polynucleotide of said INIR12
transgenic locus.
[00110] 2. The transgenic maize plant cell of embodiment la, lb, lc, id,
le, or if,
wherein said INIR12 transgenic locus comprises DNA corresponding to at least
nucleotide
number 1101 to 5830 of SEQ ID NO:1 or SEQ ID NO:46 and nucleotide number 9111
to 9360
of SEQ ID NO:1 or SEQ ID NO:46, wherein nucleotides corresponding to at least
5850 to 9090
of SEQ ID NO:1 or SEQ ID NO:46 are absent.
[00111] 3. The transgenic maize plant cell of embodiment la, lb, lc, id,
le, if, or lg,
wherein said INIR12 transgenic locus comprises the DNA molecule set forth in
SEQ ID NO:
2, 6, 29, 43, 44, 45, 47, 48, 49, or an allelic variant thereof.
[00112] 4. The transgenic maize plant cell of embodiment la, lb, lc, id,
le, or if,
wherein said INIR12 transgenic locus comprises:
(a) the DNA molecule set forth in SEQ ID NO: 3 wherein nucleotide residues
1081 to 1104
are: (i) each either absent or independently selected from a guanine, a
cytosine, an adenine
residue, or a thymine residue, with the proviso that nucleotides corresponding
to residues 1081
to 1104 of SEQ ID NO: 3 are not identical to residues 1081 to 1104 of SEQ ID
NO:1 or SEQ
ID NO:46; (ii) comprise about 2 to 8 consecutive residues of nucleotides 1081
to 1092 of SEQ
ID NO:1 or SEQ ID NO:46 and/or about 2 to 8 consecutive residues of
nucleotides 1093 to
1104 of SEQ ID NO:1 or SEQ ID NO:46, with the proviso that nucleotides
corresponding to
residues 1081 to 1104 of SEQ ID NO: 3 are not identical to residues 1081 to
1104 of SEQ ID
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NO:1 or SEQ ID NO:46; (iii) any combination of (i) and (ii); (v) are set forth
in SEQ ID NO:
7, wherein n is absent, is independently selected from A, C, G, or T,
correspond to 1 to 10
residues of nucleotides 1083 to 1092 of SEQ ID NO:1 or SEQ ID NO:46, and/or
correspond
to 1 to 10 residues of nucleotides 1093 to 1102 of SEQ ID NO:1 or SEQ ID NO:46
with the
proviso that nucleotides corresponding to nucleotide 3 to 22 of SEQ ID NO: 7
are not identical
to residues 1083 to 1102 of SEQ ID NO:1 or SEQ ID NO:46; (vi) are set forth in
SEQ ID NO:
8; wherein n is absent, is independently selected from A, C, G, or T,
correspond to 1 to 5
residues of nucleotides 1088 to1092 of SEQ ID NO:1 or SEQ ID NO:46, and/or
correspond to
1 to 5 residues of nucleotides 1093 to 1097 of SEQ ID NO:1 or SEQ ID NO:46
with the proviso
that nucleotides corresponding to residues 8 to 17 of SEQ ID NO: 8 are not
identical to residues
1088 to 1097 of SEQ ID NO:1 or SEQ ID NO:46; (vii) are set forth in SEQ ID NO:
9; wherein
n is absent, is independently selected from A, C, G, or T, correspond to 1 to
3 residues of
nucleotides 1090-1092 of SEQ ID NO:1 or SEQ ID NO:46, and/or correspond to 1
to 3 residues
of nucleotides 1093 to 1095 of SEQ ID NO:1 or SEQ ID NO:46 with the proviso
that
nucleotides corresponding to residues 1090 to 1095 of SEQ ID NO: 9 are not
identical to
nucleotides 1090 to 1095 of SEQ ID NO:1 or SEQ ID NO:46); or (viii) are set
forth in SEQ
ID NO:1 or SEQ ID NO:46, 11, 12, 13, 14, 15, 16, 17, 18, or 19 and wherein
nucleotides 5831
to 5842 of SEQ ID NO: 3 are each either absent, independently selected from a
guanine, a
cytosine, an adenine residue, or a thymine residue, comprise or consist of 1
or more nucleotides
corresponding to nucleotides 5831 to 5836 of SEQ ID NO:1 or SEQ ID
NO:46,and/or comprise
or consist of 1 or more nucleotides corresponding to nucleotides 9102 to 9107
of SEQ ID NO:1
or SEQ ID NO:46.
[00113] 5. The transgenic maize plant cell of embodiment la, lb, lc, id,
le, or if,
wherein said INIR12 transgenic locus further comprises an insertion and/or
substitution of a
DNA element comprising a cognate guide RNA recognition site (CgRRS) in a DNA
junction
polynucleotide of said INIR12 transgenic locus.
[00114] 6. The transgenic maize plant cell of embodiment lg or 5, wherein
said cognate
guide RNA recognition site (CgRRS) comprises SEQ ID NO: 26, 27, or 28, wherein
the
insertion and/or substitution is in a 5' junction polynucleotide of said
INIR12 transgenic locus
and optionally wherein the insertion and/or substitution is in a 5' junction
polynucleotide of
the INIR12 transgenic locus corresponding to at least one of nucleotides 1079
to 1098 of SEQ
ID NO:1 or SEQ ID NO:46.
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[00115] 7. The transgenic maize plant cell of embodiment 6, wherein said
CgRRS
comprises the DNA molecule set forth in SEQ ID NO: 37.
[00116] 8. The transgenic maize plant cell of embodiment la, lb, lc, id,
le, if, or lg,
wherein said INIR12 transgenic locus comprises the DNA molecule set forth in
SEQ ID NO:
2, 3, 4, 5, 6, 29, 43, 44, 45, 47, 48, 49, or an allelic variant thereof, or
wherein said MIR162
transgenic locus is set forth in SEQ ID NO:1 or SEQ ID NO:46, is present in
seed deposited at
the ATCC under accession No. PTA-8166, is present in progeny thereof, is
present in allelic
variants thereof, or is present in other variants thereof.
[00117] 9. A transgenic maize plant part comprising the maize plant cell
of any one of
embodiments la, lb, lc, id, le, if, lg, 2, 3, 4, 5, 6, 7, or 8, wherein said
maize plant part is
optionally a seed.
[00118] 10. A transgenic maize plant comprising the maize plant cell of
any one of
embodiments la, lb, lc, id, le, if, lg, 2, 3, 4, 5, 6, 7, 8, or 8.
[00119] 11. A method for obtaining a bulked population of inbred seed
comprising
selfing the transgenic maize plant of embodiment 10 and harvesting seed
comprising the
INIR12 transgenic locus from the selfed maize plant.
[00120] 12. A method of obtaining hybrid maize seed comprising crossing
the transgenic
maize plant of embodiment 10 to a second maize plant which is genetically
distinct from the
first maize plant and harvesting seed comprising the INIR12 transgenic locus
from the cross.
[00121] 13. A DNA molecule comprising SEQ ID NO: 7, 8, 9, 10, 11, 12, 13,
14, 15,
16, 17, 18, 19, 25, 37, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50-167, or an
allelic variant thereof
[00122] 14. A processed transgenic maize plant product comprising the DNA
molecule
of embodiment 13.
[00123] 15. A biological sample containing the DNA molecule of embodiment
13.
[00124] 16. A nucleic acid molecule adapted for detection of genomic DNA
comprising
the DNA molecule of embodiment 13, wherein said nucleic acid molecule
optionally comprises
a detectable label.
[00125] 17. A method of detecting a plant cell comprising the INIR12
transgenic locus
of any one of embodiments 1 a, b, c, d, e, or f to 8, comprising the step of
detecting DNA
molecule comprising SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 25, 37, 39, 40,
41, 42, 43, 44, 45, 47, 48, 49, 50-167, or an allelic variant thereof.
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[00126] 18. A method of excising the INIR12 transgenic locus from the
genome of the
maize plant cell of any one of embodiments 5, 6, 7, or 8, comprising the steps
of:
(a) contacting the edited transgenic plant genome of the plant cell of
embodiment 5, 6,
7, or 8 with: (i) an RNA dependent DNA endonuclease (RdDe); and (ii) a guide
RNA (gRNA)
capable of hybridizing to the guide RNA hybridization site of the OgRRS and
the CgRRS;
wherein the RdDe recognizes a OgRRS/gRNA and a CgRRS/gRNA hybridization
complex;
and,
(b) selecting a transgenic plant cell, transgenic plant part, or transgenic
plant wherein
the INIR12 transgenic locus flanked by the OgRRS and the CgRRS has been
excised.
[00127] 19. The method of embodiment 18, wherein the OgRRS is located in a
3'
flanking DNA junction polynucleotide and comprises SEQ ID NO: 26, 27, or 28
and wherein
the CgRRS comprises an insertion or substitution of SEQ ID NO: 26, 27, or 28
in a 5' junction
polynucleotide of said INIR12 transgenic locus.
[00128] 20. The method of embodiment 19, wherein the insertion and/or
substitution is
in a 5' junction polynucleotide of the INIR12 transgenic locus corresponding
to at least one of
nucleotides 1079 to 1098 of SEQ ID NO:1 or SEQ ID NO:46.
[00129] 21. The method of embodiment 19, wherein the CgRRS comprises the
DNA
molecule set forth in SEQ ID NO: 37.
[00130] 20a. A method of modifying a transgenic maize plant cell
comprising: obtaining
a MIR162 maize event plant cell, a representative sample of which was
deposited at the ATCC
under accession No. PTA-8166, comprising a nucleotide sequence comprising a
first ZmUbiInt
promoter, a vip3Aa19 or vip3Aa20 coding region which is operably linked to
said promoter, a
CaMV 35S terminator element which is operably linked to said vip3Aa19 or
vip3Aa20 coding
region, a second ZmUbiInt promoter and an operably linked phosphomannose
isomerase
coding region, and a nopaline synthase terminator element; and modifying said
nucleotide
sequence to eliminate functionality of said phosphomannose isomerase coding
region and/or
to substantially, essentially, or completely remove said phosphomannose
isomerase coding
region, and optionally to eliminate functionality of, or substantially,
essentially, or completely
remove, said second ZmUbiInt promoter.
[00131] 20b. A method of modifying a transgenic maize plant cell
comprising: obtaining
a MIR162 maize event plant cell, a representative sample of which was
deposited at the ATCC
under accession No. PTA-8166, comprising a nucleotide sequence comprising a
first ZmUbiInt
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promoter, a vip3Aa19 or vip3Aa20 coding region which is operably linked to
said promoter, a
CaMV 35S terminator element which is operably linked to said vip3Aa19 or
vip3Aa20 coding
region, a second ZmUbiInt promoter and an operably linked phosphomannose
isomerase
coding region, and a nopaline synthase terminator element; and modifying said
nucleotide
sequence to substantially, essentially, or completely remove said
phosphomannose isomerase
coding region, and optionally substantially, essentially, or completely remove
said second
ZmUniInt promoter.
[00132] 20c.A method of making transgenic maize plant cell comprising an
INIR12
transgenic locus comprising:
(a) contacting the transgenic plant genome of a maize MIR162 plant cell with:
(i) a first set of
gene editing molecules comprising a first site-specific nuclease which
introduces a first double
stranded DNA break in a 5' junction polynucleotide of an MIR162 transgenic
locus; and (ii) a
second set of gene editing molecules comprising a second site-specific
nuclease which
introduces a second double stranded DNA break between the CaMV35S terminator
element
and the ZmUbi promoter of said MIR162 transgenic locus which is operably
linked to DNA
encoding a phosphomannose isomerase (pmi) and a third site specific nuclease
which
introduces a third double stranded DNA break between the DNA encoding the pmi
and DNA
encoding the nopaline synthase (nos) terminator element of said MIR162
transgenic locus; and
(b) selecting a transgenic maize plant cell, transgenic maize callus, and/or a
transgenic
maize plant comprising an INIR12 transgenic locus wherein one or more
nucleotides of said
5' junction polynucleotide have been deleted and/or substituted, wherein the
first ZmUbiInt
promoter, the vip3Aa19 or vip3Aa20 coding region which is operably linked to
the first
ZmUbiInt promoter, the CaMV 35S terminator element which is operably linked to
said
vip3Aa19 or vip3Aa20 coding region, and the nos terminator element of said
MIR162
transgenic locus are present, and wherein DNA of said MIR162 transgenic locus
comprising a
second ZmUbiInt promoter and an operably linked phosphomannose isomerase
coding region
is absent, thereby making a transgenic maize plant cell comprising an INIR12
transgenic locus.
[00133] 21. The method of embodiment 20c, comprising:
(a) contacting the transgenic plant genome of a maize MIR162 plant cell with:
(i) a first set of
gene editing molecules comprising a first site-specific nuclease which
introduces a first double
stranded DNA break between nucleotide residues corresponding to nucleotide
number 1079 to
1098 of SEQ ID NO:1 or SEQ ID NO:46; and (ii) a second set of gene editing
molecules
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comprising a second site-specific nuclease which introduces a second double
stranded DNA
break between nucleotide residues corresponding to nucleotide number 5838 to
5858 of SEQ
ID NO:1 or SEQ ID NO:46 and a third site specific nuclease which introduces a
third double
stranded DNA break between nucleotide residues corresponding to nucleotide
number 9040 to
9105 of SEQ ID NO:1 or SEQ ID NO:46; and
(b) selecting a transgenic maize plant cell, transgenic maize plant callus,
and/or a
transgenic maize plant wherein one or more nucleotides corresponding to
nucleotide number
1081 to 1104 of SEQ ID NO:1 or SEQ ID NO:46 have been deleted and/or
substituted, wherein
nucleotides corresponding to at least nucleotide number 5858 to 9040 of SEQ ID
NO:1 or SEQ
ID NO:46 have been deleted and/or replaced, and wherein nucleotides
corresponding to at least
nucleotide number 1105 to 5837 of SEQ ID NO:1 or SEQ ID NO:46 are retained.
[00134] 22. The method of embodiment 20c or 21, further comprising
contacting the
transgenic plant genome of the maize MIR162 plant cell with a donor DNA
template
comprising a cognate guide RNA recognition site (CgRRS), wherein said CgRRS
optionally
comprises a polynucleotide set forth in SEQ ID NO: 26, 27, 28, or 37; and
selecting a transgenic
plant cell wherein said CgRRS has integrated into and/or replaced one or more
nucleotides
corresponding to at least one of nucleotides 1079 to 1098 of SEQ ID NO:1 or
SEQ ID NO:46.
[00135] 23. The method of any one of embodiments 20c or 21, wherein the
gene editing
molecules comprise: (i) a zinc finger nuclease; (ii) a TALEN; and/or (iii) an
RNA dependent
DNA endonuclease (RdDe) and a guide RNA.
[00136] 24. The method of embodiment 23, wherein the RNA dependent DNA
endonuclease (RdDe) comprises a Cas12a RdDe and wherein the guide RNA of said
first set
of gene editing molecules comprises SEQ ID NO: 20, the guide RNA of said
second set of
gene-editing molecules comprises SEQ ID NO: 21, and the guide RNA of said
third set of
gene-editing molecules comprises SEQ ID NO: 23.
[00137] 25. The method of any one of embodiments 20a, b, or c to 24,
further comprising
the step of regenerating transgenic maize plant callus and/or a transgenic
maize plant
comprising the modification or the INIR12 transgenic locus from said
transgenic maize plant
cell selected in step (c).
[00138] 26. The method of any one of embodiments 20a, b, or c to 25,
further comprising
the step of harvesting a transgenic maize plant seed comprising the
modification or the INIR12
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transgenic locus from the transgenic maize plant comprising the modification
or the INIR12
transgenic locus.
[00139] 27. A transgenic maize plant cell comprising a modification or an
INIR12
transgenic locus made by the method of any one of embodiments 20a, b, or c to
25.
[00140] 28. Transgenic maize plant callus comprising a modification or an
INIR12
transgenic locus made by the method of any one of embodiments 20a, b, or c to
25.
[00141] 29. A transgenic maize plant comprising a modification or an
INIR12 transgenic
locus made by the method of any one of embodiments 20a, b, or c to 25.
[00142] 30. A transgenic maize plant seed comprising a modification or an
INIR12
transgenic locus made by the method of embodiment 26.
[00143] 31. A method of using the maize plant cell of any one of
embodiments la, b, c,
d, e, f, h, or g; 2-8, or 27, the maize plant callus of embodiment 28, the
maize plant of
embodiment 10 or 29, maize plant part of embodiment 9, or maize plant seed of
embodiment
30 for collecting nucleic acid analysis data; wherein said method comprises:
(a) isolating the
nucleic acids from the maize plant cell, the maize plant callus, the maize
plant, the maize plant
part, or the maize plant seed of and analyzing said nucleic acids, and (c)
recording data based
on the analysis of the nucleic acids; wherein the nucleic acid analysis data
are optionally nucleic
acid sequence data or nucleic acid abundance data.
[00144] 32. A method of collecting nucleic acid analysis data comprising:
(a) isolating
nucleic acids from the maize plant cell of any one of embodiments la, b, c, d,
e, f, h, or g, 2-8,
or 27, the maize plant callus of embodiment 28, the maize plant of embodiment
10 or 29, maize
plant part of embodiment 9, or maize plant seed of embodiment 30; (b)
analyzing said nucleic
acids; and (c) recording data based on the analysis of the nucleic acids;
wherein the nucleic
acid analysis data are optionally nucleic acid sequence data or nucleic acid
abundance data.
[00145] 33. A method of plant breeding comprising: (a) isolating nucleic
acids from the
maize plant cell of any one of embodiments la, b, c, d, e, f, h, or g, 2-8, or
27, the maize plant
callus of embodiment 28, the maize plant of embodiment 10 or 29, maize plant
part of
embodiment 9, or maize plant seed of embodiment 30; (b) identifying one or
more nucleic acid
polymorphisms from the isolated nucleic acids; and (c) selecting a plant
having one or more of
the identified nucleic acid polymorphisms.
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Examples
[00146] Example 1. Application of a Cas12a and guide RNAs to change or
excise the
5'-T-DNA junction sequence in the MIR162 event.
[00147] The MIR162 5'- junction sequence shown in Figure 4 is flanked by
three
Cas12a recognition sequences, gRNA-1 (SEQ ID NO: 20), gRNA-2 (SEQ ID NO: 21),
and
gRNA-3 (SEQ ID NO: 22) that can be used to modify some of the 5' junction
sequence or
eliminate most of it. There are four possible iterations of this approach. The
first two depend
on gRNA-1 and gRNA-3 alone to disrupt the MIR162 5'-junction sequence. The
second two
combine gRNA-2 with either gRNA-1 or gRNA-3 to eliminate most of the MIR162
junction
sequence. In certain instances, gRNA-1 (SEQ ID NO: 20) is used to modify the
5' DNA
junction polynucleotide and obtain a modified 5' junction polynucleotide
comprising SEQ ID
NO: 39 or 40.
[00148] The Cas12a nuclease and the single or combined gRNAs are
introduced into the
MIR162 event. This can be accomplished in different ways that are familiar to
those with
ordinary skill in the art. The first is to encode expression of the Cas12a
nuclease and gRNA(s)
on a T-DNA and transform it into the MIR162 event via Agrobacterium-mediated
transformation. Alternatively, the T-DNA can be transformed into any
convenient maize line,
and then crossed with the MIR162 event to combine the Cas12a ribonucleoprotein
expressing
T-DNA with the MIR162 event. The Cas12a nuclease and gRNAs can also be
assembled in
vitro then delivered to MIR162 explants as ribonucleoprotein complexes using a
biolistic
approach (Svitashev et al., 2016; doi: 10.1038/nc0mm513274). Also, a plasmid
encoding a
Cas12a nuclease and the gRNA(s) can be delivered to MIR162 explants using a
biolistic
approach. This will produce plant cells that have a high likelihood of
incurring mutations that
disrupt the MIR162 junction sequence. To use the Agrobacterium approach a
binary vector that
contains a strong constitutive expression cassette like the ZmUbi 1
promoter::ZmUbi 1
terminator driving Cas12a, a PolII or PolIII gene cassette driving the Cas12a
gRNA(s) and a
CaMV 355:PAT:NOS or other suitable plant selectable marker is constructed. An
expression
cassette driving a fluorescent protein like mScarlet may also be useful to the
plant
transformation process. Constructs are transformed into Agrobacterium strain
LBA4404.
[00149] Maize transformations are performed based on published methods
(Ishida et. al,
Nature Protocols 2007; 2, 1614-1621). Briefly, immature embryos from inbred
line GIBE0104,
approximately 1.8-2.2 mm in size, are isolated from surface sterilized ears 10-
14 days after
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pollination. Embryos are placed in an Agrobacterium suspension made with
infection medium
at a concentration of OD 600=1Ø Acetosyringone (20011M) is added to the
infection medium
at the time of use. Embryos and Agrobacterium are placed on a rocker shaker at
slow speed for
15 minutes. Embryos are then poured onto the surface of a plate of co-culture
medium. Excess
liquid media is removed by tilting the plate and drawing off all liquid with a
pipette. Embryos
are flipped as necessary to maintain a scutelum up orientation. Co-culture
plates are placed in
a box with a lid and cultured in the dark at 22 C for 3 days. Embryos are
then transferred to
resting medium, maintaining the scutellum up orientation. Embryos remain on
resting medium
for 7 days at 27-28 C. Embryos that produce callus are transferred to
Selection 1 medium with
7.5 mg/L phosphinothricin (PPT) and cultured for an additional 7 days.
Callused embryos are
placed on Selection 2 medium with 10 mg/L PPT and cultured for 14 days at 27-
28 C.
Growing calli resistant to the selection agent are transferred to Pre-
Regeneration media with
mg/L PPT to initiate shoot development. Calli remain on Pre-Regeneration media
for 7
days. Calli beginning to initiate shoots are transferred to Regeneration
medium with 7.5 mg/L
PPT in Phytatrays and cultured in light at 27-28 C. Shoots that reach the top
of the Phytatray
with intact roots are transferred to Shoot Elongation medium prior to
transplant into soil and
gradual acclimatization to greenhouse conditions.
[00150] When a sufficient amount of viable tissue is obtained, it can be
screened for
mutations at the MIR162 junction sequence, using a PCR-based approach. One way
to screen
is to design DNA oligonucleotide primers that flank and amplify the MIR162
junction plus
surrounding sequence. For example, the primers (5'-tttgcatcattggtgtcatcagttttt-
3'; SEQ ID NO:
30) and (5'-tttcccgccttcagtttaaactatcag-3'; SEQ ID NO: 31) will produce a ¨310
bp product that
can be analyzed for edits at the target site. The size of this product will
vary based on the nature
of the edit. Amplicons can be sequenced directly using an amplicon sequencing
approach or
ligated to a convenient plasmid vector for Sanger sequencing. Those plants in
which the
MIR162 5'-junction sequence is disrupted are selected and grown to maturity.
The DNA
encoding the Cas12a reagents can be segregated away from the modified junction
sequence in
a subsequent generation.
[00151] Example 2. Insertion of a CgRRS element in the 5'-junction of the
MIR162
event.
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[00152] This example describes the construction of plant expression
vectors for
Agrobacterium mediated maize transformation. Two plant gene expression vectors
are
prepared. Plant expression cassettes for expressing a Bacteriophage lambda
exonuclease, a
bacteriophage lambda beta SSAP protein, and an E. coli SSB are constructed
essentially as set
forth in US Patent Application Publication 20200407754, which is incorporated
herein by
reference in its entirety. A DNA sequence encoding a tobacco c2 nuclear
localization signal
(NLS) is fused in-frame to the DNA sequences encoding the exonuclease, the
bacteriophage
lambda beta SSAP protein, and the E. coli SSB to provide a DNA sequence
encoding the c2
NLS-Exo, c2 NLS lambda beta SSAP, and c2 NLS-SSB fusion proteins that are set
forth in
SEQ ID NO: 135, SEQ ID NO: 134, and SEQ ID NO: 133 of US Patent Application
Publication
20200407754, respectively, and incorporated herein by reference in its
entirety. DNA
sequences encoding the c2 NLS-Exo, c2 NLS lambda beta SSAP, and c2NLS-SSB
fusion
proteins are operably linked to a OsUBIL ZmUBIL OsACT promoter and a OsUbil,
ZmUBIL
OsACT polyadenylation site respectively, to provide the exonuclease, SSAP, and
SSB plant
expression cassettes.
[00153] A donor DNA template sequence (SEQ ID NO: 32) that targets the 5'-
T-DNA
junction of the MIR162 event for insertion of a 27 base pair heterologous
sequence, that is
identical to a Cas12a recognition site at the 3'-junction of the MIR162 T-DNA
insert, by HDR
is constructed. The donor DNA template sequence includes a replacement
template with
desired insertion region (27 base pair long sequence of SEQ ID NO: 27) flanked
on both sides
by homology arms about 500-635 bp in length. The homology arms match (i.e.,
are
homologous to) gDNA (genomic DNA) regions flanking the target gDNA insertion
site. The
replacement template region comprising the donor DNA template is flanked at
each end by
DNA sequences identical to the MIR162 5' polynucleotide sequence recognized by
an RNA-
guided nuclease and one or more gRNA(s) (e.g. gRNAs comprising SEQ ID NO: 20,
21, and
22). In certain cases, a deletion is made in the targeted MIR162 5'
polynucleotide sequence
(e.g., using gRNAs comprising SEQ ID NO: 20 and 21 in combination or by using
gRNAs
comprising SEQ ID NO: 21 and 22 in combination).
[00154] A plant expression cassette that provides for expression of the
RNA-guided
sequence-specific Cas12a endonuclease is constructed. A plant expression
cassette that
provides for expression of a guide RNA complementary to sequences adjacent to
the insertion
site (e.g. gRNAs comprising SEQ ID NO: 20, 21, and 22) is constructed.
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An Agrobacterium superbinary plasmid transformation vector containing a
cassette that
provides for the expression of the phosphinothricin N-
acetyltransferasesynthase (PAT) protein
is constructed. Once the cassettes, donor sequence and Agrobacterium
superbinary plasmid
transformation vector are constructed, they were combined to generate two
maize
transformation plasmids.
[00155] A maize transformation plasmid is constructed with the PAT
cassette, the RNA-
guided sequence-specific endonuclease cassette, the guide RNA cassette, and
the MIR162 5' -
junction polynucleotide donor DNA template sequence into the Agrobacterium
superbinary
plasmid transformation vector (the control vector).
[00156] A maize transformation plasmid is constructed with the PAT
cassette, the RNA-
guided sequence-specific endonuclease cassette, the guide RNA cassette, the
SSB cassette, the
lambda beta SSAP cassette, the Exo cassette, and the MIR162 5'- junction
polynucleotide
donor DNA template into the Agrobacterium superbinary plasmid transformation
vector (the
lambda red vector).
[00157] All constructs are delivered from superbinary vectors
in Agrobacterium strain LBA4404.
[00158] Maize transformations are performed based on published methods
(Ishida
et. al, Nature Protocols 2007; 2, 1614-1621). Briefly, immature embryos from
inbred line
GIBE0104, approximately 1.8-2.2 mm in size, are isolated from surface
sterilized ears 10-
14 days after pollination. Embryos are placed in an Agrobacterium suspension
made with
infection medium at a concentration of OD 600=1Ø Acetosyringone (20011M) is
added to
the infection medium at the time of use. Embryos and Agrobacterium are placed
on a
rocker shaker at slow speed for 15 minutes. Embryos are then poured onto the
surface of
a plate of co-culture medium. Excess liquid media is removed by tilting the
plate and
drawing off all liquid with a pipette. Embryos are flipped as necessary to
maintain a
scutelum up orientation. Co-culture plates are placed in a box with a lid and
cultured in
the dark at 22 C for 3 days. Embryos are then transferred to resting medium,
maintaining
the scutellum up orientation. Embryos remain on resting medium for 7 days at
27-28 C.
Embryos that produced callus are transferred to Selection 1 medium with 7.5
mg/L
phosphinothricin (PPT) and cultured for an additional 7 days. Callused embryos
are placed
on Selection 2 medium with 10 mg/L PPT and cultured for 14 days at 27-28 C.
Growing
calli resistant to the selection agent are transferred to Pre-Regeneration
media with 10
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mg/L PPT to initiate shoot development. Calli remained on Pre-Regeneration
media for 7
days. Calli beginning to initiate shoots are transferred to Regeneration
medium with 7.5
mg/L PPT in Phytatrays and cultured in light at 27-28 C. Shoots that reached
the top of
the Phytatray with intact roots are isolated into Shoot Elongation medium
prior to
transplant into soil and gradual acclimatization to greenhouse conditions.
[00159] When a sufficient amount of viable tissue is obtained, it can be
screened
for insertion at the MIR162 junction sequence, using a PCR-based approach. The
PCR
primer on the 5'-end can be 5'-ttttatgtattatttggtccctaca-3' (SEQ ID NO: 33)
and the PCR
primer on the 3'-end is 5'-gtcgacggcgtttaacaggctggca-3' (SEQ ID NO: 34). These
primers
that flank donor DNA homology arms are used to amplify the MIR162 5'-junction
sequence. The correct donor sequence insertion will produce a 1579 bp product.
Amplicons can be sequenced directly using an amplicon sequencing approach or
ligated
to a convenient plasmid vector for Sanger sequencing. Those plants in which
the MIR162
5' junction polynucleotide sequence now contains the intended CgRRS (e.g.,
Cas12a
recognition sequence in SEQ ID NO: 37) are selected and grown to maturity. The
T-DNA
encoding the Cas12a reagents can be segregated away from the modified junction
sequence in a subsequent generation. The resultant INIR12 transgenic locus
comprising
the CgRRS and OgRRS (e.g. which each comprise SEQ ID NO: 27 and an operably
linked
PAM site) can be excised using Cas12a and a suitable gRNA which hybridizes to
DNA
comprising SEQ ID NO: 27 at both the OgRRS and the CgRRS. An example of an
INIR12
locus comprising the intended CgRRS in SEQ ID NO: 37 is provided as SEQ ID NO:
44.
Another example of an INIR12 locus comprising the intended CgRRS in SEQ ID NO:
37
is provided as SEQ ID NO: 47 and is illustrated in Figure 6.
[00160] Example 3. Deletion of the MIR162 PMI gene cassette.
[00161] The ZmUbil::PMI coding sequence in MIR162 transgenic maize
performs no
useful function with respect to field productivity. It can be removed using a
Cas12a-mediated
genomic DNA deletion approach. The procedure calls for creating an
Agrobacterium
transformation vector encoding the Cas12a nuclease, the MIR162 PMI 5' guide
RNA (5'-
taattcctaaaaccaaaatccag-3'; SEQ ID NO: 23), the MIR162 PMI 3' guide RNA (5'-
ttgccaaatgtttgaacgatctg-3' ; SEQ ID NO: 24), and a plant selectable marker
gene.
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[00162] A
binary vector that contains a strong constitutive expression cassette like the
ZmUbil promoter::ZmUbil terminator driving Cas12a, a PolII or PolIII gene
cassette driving
the Cas12a gRNAs and a CaMV 35S:PAT:NOS or other suitable plant selectable
marker is
constructed. An expression cassette driving a fluorescent protein like
mScarlet may also be
useful to the plant transformation process and included in the binary vector.
[00163] The
aforementioned binary vector is transformed into maize using the procedure
essentially as outlined in Example 1. The regenerated plants can be screened
with the primer
set below to identify individuals that have lost the ZmUbil::PMI fragment. The
primers span
3820 bases in the intact insert. If both cuts occur and the ends are ligated
together, this will
produce a ¨555 bp amplicon. This is verified by DNA sequence analysis. The
primer set
includes 162-PMI-ampseq-5' (5'-ggcaacaacctgtacggcggcccga-3'; SEQ ID NO: 35)
and 162-
PMI-ampseq-3' (5'-gttgccttcagaccatggcggacgt-3'; SEQ ID NO: 36).
[00164]
Example 4. Introduction of a CgRRS into an INIR12 maize plant comprising a
deletion of the MIR162 ZmUbil::PMI fragment
[00165]
Maize plants comprising the deletion of the MIR162 ZmUbil:PMI fragment are
subjected to the procedures for integration of the SEQ ID NO: 32 donor DNA
template set
forth in Example 2 to provide for a resultant INIR12 transgenic locus
comprising the CgRRS
and OgRRS (e.g. which each comprise SEQ ID NO: 27 and an operably linked PAM
site)
where the ZmUbil::PMI fragment is absent. This resultant INIR12 transgenic
locus can be
excised using Cas12a and a suitable gRNA which hybridizes to DNA comprising
SEQ ID NO:
27 at both the OgRRS and the CgRRS. An example of a INIR12 transgenic locus
comprising
the deletion of the MIR162 ZmUbi 1 :PMI fragment, the CgRRS sequence, and the
OgRRS
sequence (e.g. which each comprise SEQ ID NO: 27 and an operably linked PAM
site) is set
forth in SEQ ID NO: 48.
[00166]
Example 5. Application of Cas12a and SEQ ID NO: 20 and SEQ ID NO: 22
guide RNAs to change or excise the 5' -T-DNA junction sequence in the MIR162
event.
[00167]
Cas12a and SEQ ID NO: 20 and SEQ ID NO: 22 guide RNAs to are used to
excise portions of a 5' junction sequence in the MIR162 event as follows.
[00168]
Maize protoplasts were prepared essentially as described in a preparation
protocol modified from one publicly available at
molbio[dot]mgh[dot]harvard.edu/sheenweb/protocols reg[dot]html. Healthy leaf
tissue minus
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the midveins from the third leaf of 10 day old plants were sliced into 0.5 mm
segments using
a fresh scalpel in Enzyme Solution (0.6 M mannitol, 10 mM IVIES, 20 mM KC1, 20
mM CaCl2,
0.1% BSA, 1.5% Cellulase RS, 0.3% Macerozyme RO at pH 5.7 and sterilized using
a 0.2
micron filter). The tissued was vacuum infiltrated for 30 minutes, and up to 8
leaves were
processed in 20 mL Enzyme Solution. Continue digestion for 2.5 hours at 26 C
on shaking
incubator at 60 rpm. Samples were sealed with parafilm and incubated in the
dark for 2.5 hours
at 26 C while shaking at 60 rpm. The Enzyme solution containing protoplasts
was passed
through a 40 p.m sterile cell strainer into a conical 50m1 falcon tube. The
dish was rinsed with
mL PVB I solution (0.6 M mannitol, 10 mM IVIES, 20 mM KC1, 20 mM CaCl2, 0.1%
BSA
at pH 5.7 and sterilized using a 0.2 micron filter) which was also passed
through the 40 p.m
sterile cell strainer. The protoplast solution was transferred to a conical
round bottom centrifuge
tube and gently centrifuged at 200 x g for 2 minutes in a swinging bucket
rotor. The protoplasts
were gently resuspended in 20 mL PVB I solution then centrifuged as before.
This wash step
was repeated once more, then the protoplasts were suspended in PVB I solution
at 1000000
protoplasts/mL.
[00169] Each plasmid vector encoding genome editing reagents and the
fluorescent
marker mScarlet (see Sample column in Table 1) was transfected into
protoplasts by adding
approximately 2 x 1012 copies of plasmid DNA (in 20 tL TE buffer) to 200 tL of
cells
(-250000). Then 220 tL of PEG solution (200 mM mannitol, 100 mM CaCl2, 40%
PEG) was
added and the mixture was incubated for 5 minutes at room temperature. Then 1
mL of W5
solution (10 mM MES, 154 mM NaCl, 125 mM CaCl2, 5 mM KC1, 0.1% BSA at pH 5.8
and
sterilized using a 0.2 micron filter) was gently added, and the protoplast
solution was
centrifuged at 200 x g for 3 minutes. The protoplast pellet was gently
resuspended in 1.7 mL
W5 solution and centrifuged cells at 200 x g for 2 minutes. The protoplasts
were resuspended
in W5 buffer, then transferred to a 24 well cell culture plate, at 50000 cells
in 5001.iL W5 buffer
per well. The plates were sealed with Parafilm, wrapped in aluminum foil, and
incubated
overnight at 28 in an incubator with zero agitation.
[00170] Approximately 44 hours after transfection viable protoplasts were
assessed by
Fluorescein Diacetate (FDA) staining and dead cells were assessed by
SytoxGreen staining
using standard procedures. The transfection efficiency is the ratio of viable
protoplasts to
mScarlet positive protoplasts. Results for a typical experiment are in Table
1. Flow cytometry
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was used to isolate viable transfected cells expressing mScarlet. Identical
cell samples were
pooled and stored at -80 C until DNA extraction.
[00171] Table 1. Typical Transfection Analysis
Sample Type Protoplasts of of
FDA+ events mScarlet+
% of
% %
Events Events Events
FDA
total total
B104
with TE
9273 47.0 0 0 0 0
buffer no wt
fda control
B104
with TE wt 9228 51.6 2761 29.9 0 0
buffer control
MIR162
with TE
9299 68.7 0 0 0 0
buffer no buffer
fda control
MIR162
with TE
9340 66.3 1368 14.6 1 0.0
buffer no buffer
fda control
MIR162
with
Cas12a 9237 67.5 2 0.0 2
100.0
sample 1 vector
no FDA control
MIR162
with
9408 69.1 1839 19.5 329
17.9
Cas12a vector
sample 1 control
MIR162
with
9250 64.5 1474 15.9 320
21.7
Cas12a vector
sample 2 control
MIR162
with
SEQ ID
9266 67.3 1435 15.5 238
16.6
NO: 22
vector
sample 1 Edit 1
MIR162
with
SEQ ID
9081 64.4 1619 17.8 251
15.5
NO: 22
vector
sample 2 Edit 1
MIR162
with 9275 66.1 1547 16.7 269
17.4
SEQ ID Edit 1
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NO: 22
vector
sample 3
MIR162
with
SEQ ID
NO 20 9292 67.9 1417 15.2 291
20.5
:
vector
sample 1 Edit 2
MIR162
with
SEQ ID
NO 20 9364 64.9 1404 15.0 242
17.2
:
vector
sample 2 Edit 2
MIR162
with
SEQ ID
NO 20 9320 65.6 1457 15.6 204
14.0
:
vector
sample 3 Edit 2
[00172] The gDNA isolation procedure combines the Qiagen DNeasy plant
mini kit
protocol with a magnetic bead based nucleic acid clean-up protocol similar to
the MAGBIO
(Gaithersburg. MD, USA) HighPrepTM PCR Clean-up System. Added 400 [IL buffer
AP1 and
4 [IL RNase A to each sample, thoroughly vortexed and incubate at 65 C for 10
minutes. The
tubes were inverted 2-3 times during incubation. Added 130 [IL buffer P3,
mixed and incubated
for 5 minutes on ice. Centrifuged the lysate for 5 minutes at 20000 x g, then
added the lysate
into a Qiagen QIAshredder spin column and centrifuged for 2 minutes at 20000 x
g.
[00173] A U-bottom 96 well plate was prepared by adding 50 [IL resuspended
magnetic
beads per well. Then 50 [IL plant leaf lysate was added to the beads and mixed
by pipetting.
The remaining lysate was transferred to a new 2 mL Eppendorf tube, mixed with
1.5 volume
of AW1 buffer and stored at -20 C. If necessary this sample can be used to
extracted gDNA
according to the Qiagen DNeasy (ID plant mini kit protocol (starting at step
7). The bead mixture
was incubated for 5 minutes at room temperature, then the U-bottom plate was
placed on a
magnetic separation device for 3 minutes or until the solution cleared
completely. The
supernatant was removed and discarded. The beads were washed by adding 200 [IL
of 80%
ethanol and incubating for 30 seconds on the magnetic separation device. The
supernatant
removed and the 80% ethanol wash was repeated. The beads were air dried at
room temperature
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on the magnetic separation device. The gDNA was eluted by adding in 50 [IL EB
and
incubating for 2 minutes at room temperature.
[00174] The plate was placed on the magnetic separation device for 3
minutes or until
the solution cleared completely. The supernatant was transferred to a new 96
well plate and
stored at either 20 C (long term) or at 4 C (short time). Typically 1-2 [IL is
suitable template
for PCR.
[00175] The amplicons were produced in 50 [IL PCR reaction mixtures
containing 50]
[IL Q5 High-Fidelity 5X buffer, 1 [IL 10 mM dNTP mix, 0.5 [IL Q5 polymerase,
2.5 [IL 101.tM
forward primer, 2.5 [IL 10 1.tM reverse primer and 5 [IL template DNA. The
forward PCR
primer for amplicon production is set forth in SEQ ID NO: 168 and the reverse
PCT primer for
amplicon production is set forth in SEQ ID NO: 169. The thermocycling program
was 98 C
for 30 seconds followed by 37 cycles of 98 C for 10 seconds, 63 C for 30
seconds and 72 C
for 30 seconds. The final extension was 72 C for 2 minutes. Products (10 [IL)
were analyzed
on a 2% TAE agarose gel. Each reaction produced a single dominant band at the
expected size
(about 386 nucleotides). The remaining reaction products were pooled (approx.
2 x 35 ilL) and
400 tL PB buffer (Qiagen) was added. The reaction products were isolated using
the Qiagen
QIAquick PCR purification kit and each amplicon was eluted in 38 [IL EB. The
DNA
concentration was quantified and adjusted to 20 ng/pL. Amplicon sequencing
data were
generated and analyzed by standard procedures.
[00176] Results of the SEQ ID NO: 20 gRNA mediated deletions of the 5'
junction
polynucleotide of the MIR162 locus are provided in SEQ ID NO: 50-112, which
correspond to
the region located between nucleotides 920 to 1240 in the MIR162 locus
sequence of SEQ ID
NO: 46. Results of the SEQ ID NO: 22 gRNA mediated deletions of the 5'
junction
polynucleotide in the MIR162 locus are provided in SEQ ID NO: 113-167, which
correspond
to the region located between nucleotides 920 to 1240 in the MIR162 locus
sequence of SEQ
ID NO: 46. The amplicon sequence set forth in SEQ ID NO: 112 shows the
deletion of the
TAGT sequence located at position 1095 to 1098 of SEQ ID NO: 46. A full length
sequence
of an INIR12 locus comprising the deletion shown in the SEQ ID NO: 112
amplicon sequence
is shown in SEQ ID NO: 49.
[00177] The breadth and scope of the present disclosure should not be
limited by any of
the above-described embodiments.
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