Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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INHT26 TRANSGENIC SOYBEAN
REFERENCE TO SEQUENCE LISTING SUBMITTED
ELECTRONICALLY
100011 The sequence listing contained in the file named "10087W01
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 soybean event which confers
tolerance to
glyphosate, glufosinate, 2,4-dichlorophenoxyacetic acid and pyridyloxyacetate
herbicides is
the DA544406-6 transgenic soybean event disclosed in U.S. Patent No.
9,540,655.
DA544406-6 transgenic soybean plants express a 2mepsps protein which can
confer tolerance
to glyphosate, a phosphinotricin acetyl transferase (PAT) protein which
confers tolerance to
the herbicide glufosinate, and a aryloxyalkanoate dioxygenase (AAD-12) protein
which
confers tolerance to 2,4-dichlorophenoxyacetic acid and pyridyloxyacetate
herbicides.
[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
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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 soybean plant cells comprising an INHT26 transgenic locus
comprising an
originator guide RNA recognition site (OgRRS) in a first DNA junction
polynucleotide of a
DA544406-6 transgenic locus and a cognate guide RNA recognition site (CgRRS)
in a second
DNA junction polynucleotide of the DA544406-6 transgenic locus are provided.
Transgenic
soybean plant cells comprising an INHT26 transgenic locus comprising an
insertion and/or
substitution in a DNA junction polynucleotide of a DA544406-6 transgenic locus
of DNA
comprising a cognate guide RNA recognition site (CgRRS) are provided. In
certain
embodiments, the DA544406-6 transgenic locus is set forth in SEQ ID NO:1, is
present in seed
deposited at the ATCC under accession No. PTA-11336 is present in progeny
thereof, is
present in allelic variants thereof, or is present in other variants thereof.
INHT26 transgenic
soybean plant cells, transgenic soybean plant seeds, and transgenic soybean
plants all
comprising a transgenic locus set forth in SEQ ID NO: 14 are provided.
Transgenic soybean
plant parts including seeds and transgenic soybean plants comprising the
soybean plant cells
are also provided.
[0006] Methods for obtaining a bulked population of inbred seed comprising
selfing the
aforementioned transgenic soybean plants and harvesting seed comprising the
INHT26
transgenic locus from the selfed soybean plant are also provided.
[0007] Methods of obtaining hybrid soybean seed comprising crossing the
aforementioned
transgenic soybean plants to a second soybean plant which is genetically
distinct from the first
soybean plant and harvesting seed comprising the INHT26 transgenic locus from
the cross are
provided. Methods for obtaining a bulked population of seed comprising selfing
a transgenic
soybean plant of comprising SEQ ID NO: 14 and harvesting transgenic seed
comprising the
transgenic locus set forth in SEQ ID NO: 14 are provided.
[0008] A DNA molecule comprising SEQ ID NO: 14, 16, 17, or an allelic variant
thereof is
provided. Processed transgenic soybean plant products and biological samples
comprising the
DNA molecules are provided. Nucleic acid molecules adapted for detection of
genomic DNA
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comprising the DNA molecules, wherein said nucleic acid molecule optionally
comprises a
detectable label are provided. Methods of detecting a soybean plant cell
comprising the
INHT26 transgenic locus of any one of claims 1 to 3, comprising the step of
detecting a DNA
molecule comprising SEQ ID NO: 14, 16, or 17 are provided.
[0009] Methods of excising the INHT26 transgenic locus from the genome of the
aforementioned soybean plant cells comprising the steps of:(a) contacting the
edited transgenic
plant genome of the plant cell 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 INHT26 transgenic locus flanked by the OgRRS and
the CgRRS
has been excised.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
100101 Figure 1A-D shows a sequence (SEQ ID NO: 1) of the DA544406-6
event transgenic locus including the endogenous genomic DNA (uppercase),
transgenic insert
DNA (lowercase) and 5' and 3' junction sequences flanking the transgenic
insert DNA. The
OgRRS sequence comprising the Protospacer Adjacent Motif (PAM) site (TTTA) and
gRNA
hybridization site (i.e., protospacer sequence; SEQ ID NO: 19) in the genomic
DNA of the 5'
junction sequence is shown in bold and underlined. The PAM sites and gRNA
hybridization
sites for the Guide-2 (SEQ ID NO: 5), Guide-3 (SEQ ID NO: 6), and Guide-5 (SEQ
ID NO: 8)
gRNAs which are located in or span the 3' junction polynucleotide sequence are
in italics and
double underlined. The Guide-2 and Guide-3 gRNAs are directed to transgenic
DNA located
5' to the transgene/soybean genomic DNA junction. The PAM site and gRNA
hybridization
site for the Guide-5 gRNA span the transgene/soybean genomic DNA junction of
the 3'
junction polynucleotide.
[0011] Figure 2A-D shows a sequence (SEQ ID NO: 14) of the INHT26
transgenic locus including the endogenous genomic DNA (uppercase) and
transgenic insert
DNA (lowercase) as well as the 5' and 3' junction sequences flanking the
inserted transgenic
DNA. The OgRRS sequence comprising the PAM site (TTTA) and gRNA hybridization
site
(i.e., protospacer sequence; SEQ ID NO: 19) in the genomic DNA of the 5'
junction sequence
is shown in bold and underlined. A CgRRS comprising the PAM site (TTTA) and
gRNA
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hybridization site (i.e., protospacer sequence; SEQ ID NO: 19) located in the
endogenous
genomic DNA of the 3' junction polynucleotide is also shown in bold and
underlined. The
CgRRS as depicted can be introduced into the 3' junction polynucleotide as
shown by using
the Guide-5 gRNA hybridization site of SEQ ID NO: 8, a suitable Cas RdDe
(e.g., a Cas12a
nuclease of SEQ ID NO: 15), and the donor DNA template of SEQ ID NO: 11. The
INHT26
transgenic locus can be excised with a single guide RNA which hybridizes to
the SEQ ID NO:
19 gRNA hybridization site and a suitable Cas RdDe (e.g., a Cas12a nuclease of
SEQ ID NO:
15) which will cleave DNA in both the OgRRS which flanks the 5' end of the
INHT26
transgenic locus and the OgRRS which flanks the 3' end of the INHT26
transgenic locus.
[0012] Figure 3 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 3, "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") with genome editing molecules.
[0013] Figure 4A, B, C. Figure 4A 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 2nd junction polynucleotide (bottom). Figure 4B 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 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 4C 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 the
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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 4C, 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 excised and non-transgenic DNA present in the untransformed plant
chromosome is at least
partially restored.
DETAILED DESCRIPTION
[0014] 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.
[0015] Where a term is provided in the singular, the inventors also
contemplate embodiments
described by the plural of that term.
[0016] 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.
[0017] 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.
[0018] 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).
[0019] 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,
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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.
[0020] The term "backcross", as used herein, refers to crossing an F 1 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.
[0021] 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 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.
[0022] 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) with respect to the reference
polynucleotide sequence
(e.g., SEQ ID NO: 1) 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).
[0023] As used herein, the terms "Cpfl" and "Cas12a" are used interchangeably
to refer to the
same RNA dependent DNA endonuclease (RdDe). A Cas12a protein provided herein
includes
the protein of SEQ ID NO: 15.
[0024] 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
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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.
100251 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 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.
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[0026] 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.
[0027] As used herein, the term "DAS44406-6" is used to refer to any of a
transgenic soybean
locus, transgenic soybean plants and parts thereof including seed set forth in
US Patent No.
9,540,655, which is incorporated herein by reference in its entirety.
Representative DAS44406-
6 transgenic soybean seed have been deposited with American Type Culture
Collection
(ATCC, Manassas, Va. 20110-2209 USA) under Accession No. PTA-11336. DA544406-6
transgenic loci include loci having the sequence of SEQ ID NO:1, the sequence
of the
DA544406-6 locus in the deposited seed of Accession No. PTA-11336 and any
progeny
thereof, as well as allelic variants and other variants of SEQ ID NO: 1.
[0028] 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.
[0029] 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 F 1
progeny of a cross
between two distinct elite inbred or doubled haploid plant lines.
[0030] 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' junction polynucleotide and confer one or more useful traits
including herbicide
tolerance, insect resistance, male sterility, and the like.
[0031] 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.
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[0032] 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.
[0033] As used herein, the term "Fl" refers to any offspring of a cross
between two genetically
unlike individuals.
[0034] 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.
[0035] 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.
[0036] As used herein, the term "INHT26" is used to refer either individually
collectively to
items that include any or all of the DAS44406-6 transgenic soybean loci which
have been
modified as disclosed herein, modified DAS44406-6 transgenic soybean plants
and parts
thereof including seed, and DNA obtained therefrom.
[0037] The term "isolated" as used herein means having been removed from its
natural
environment.
[0038] 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.
[0039] 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
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progeny line obtained from the initial transgenic event. Examples of
introduced transgenes
include exogenous transgenes which are inserted in a resident original
transgenic locus.
[0040] 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.
[0041] 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.
[0042] 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) markers or isozyme markers or combinations of the markers
described herein
which defines a specific genetic and chromosomal location.
[0043] 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).
[0044] The term "offspring", as used herein, refers to any progeny generation
resulting from
crossing, selfing, or other propagation technique.
[0045] 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
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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 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.
[0046] 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, 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.
[0047] 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.
[0048] 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.
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[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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 (i.e.,
protospacer
sequence). 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).
[0054] 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 (i.e., protospacer sequence), 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
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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).
[0055] 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 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.
[0056] 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).
[0057] 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.
[0058] 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 soybean and soybean 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). Provided
herein are methods
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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). Also provided are plant genomes containing modified
transgenic loci
which can be selectively excised with a single gRNA molecule. Such modified
transgenic loci
comprise an originator guide RNA recognition site (OgRRS) which is identified
in non-
transgenic DNA 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 with a
single guide RNA. An originator guide RNA recognition site (OgRRS) comprises
endogenous
DNA found in untransformed plants and in endogenous non-transgenic DNA of
junction
polynucleotides of transgenic plants containing a modified or unmodified
transgenic locus. The
OgRRS located in non-transgenic DNA 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
endogenous non-transgenic DNA found in junction sequences of transgenic plants
containing
an unmodified transgenic locus. Also provided are unique transgenic locus
excision sites
created by excision of such modified transgenic loci, DNA molecules comprising
the modified
transgenic loci, unique transgenic locus excision sites and/or plants
comprising the same,
biological samples containing the DNA, nucleic acid markers adapted for
detecting the DNA
molecules, and related methods of identifying the elite crop plants comprising
unique
transgenic locus excision sites.
[0059] 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 3. In certain embodiments, INHT26
transgenic loci provided
here are characterized by polynucleotide sequences that can facilitate as
necessary the removal
of the INHT26 transgenic loci from the genome. Useful applications of such
INHT26
transgenic loci and related methods of making include targeted excision of a
INHT26
transgenic locus or portion thereof in certain breeding lines to facilitate
recovery of germplasm
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with subsets of transgenic traits tailored for specific geographic locations
and/or grower
preferences. Other useful applications of such INHT26 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, soybean genomes containing INHT26
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 INHT26 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 of junction
polynucleotides of transgenic plants containing a modified or unmodified
transgenic locus. In
certain embodiments, 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. An example of OgRRS
polynucleotide
sequences in or near a 5' junction polynucleotide in an DA544406-6 transgenic
locus include
SEQ ID NO: 18, which is shown in bold and underlined in Figure 1. 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 4C and as
elsewhere
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described herein. A donor DNA template for introducing the SEQ ID NO: 18 OgRRS
into the
3' junction polynucleotide of an DA544406-6 locus includes the donor DNA
template
comprising SEQ ID NO: 11. Double stranded breaks in a 3' junction
polynucleotide of SEQ
ID NO: 1 can be introduced with the Guide-1, 2, 3, 4, and/or 5 gRNAs, which
are respectively
encoded by SEQ ID NO: 4, 5, 6, 7, and/or 8, and a Cas12a nuclease. In certain
embodiments,
double stranded breaks in a 3' junction polynucleotide of SEQ ID NO: 1 can be
introduced
with the Guide-1 or 2 gRNAs and any one of the Guide-3, 4, and/or 5 gRNAs and
a Cas12a
nuclease (e.g., a Cas12a nuclease of SEQ ID NO: 15). Integration of the SEQ ID
NO: 11 donor
DNA template comprising the CgRRS into the 3' junction polynucleotide of an
DA544406-6
locus at the double stranded breaks introduced by the gRNAs encoded by SEQ ID
NO: 4, 5, 6,
7, and/or 8 and a Cas12a nuclease can provide an INHT26 locus comprising the
CgRRS
sequence set forth in SEQ ID NO: 14. A subsequence comprising the CgRRS which
is located
in the 3' junction polynucleotide of the INHT26 transgenic locus is set forth
in SEQ ID NO:
17. Double stranded breaks in a 3' junction polynucleotide of SEQ ID NO: 1 can
be introduced
with gRNAs encoded by SEQ ID NO: 8 and a Cas12a nuclease. A donor DNA template
of
SEQ ID NO: 11 or the equivalent thereof having longer or shorter homology arms
can be used
to obtain the CgRRS insertion in the 3' junction polynucleotide that is set
forth in SEQ ID NO:
17. An INHT26 transgenic locus containing this CgRRS insertion is set forth in
SEQ ID NO:
14.
[0060] Also provided herein are allelic variants of any of the INHT26
transgenic loci or DNA
molecules provided herein. In certain embodiments, such allelic variants of
INHT26 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,
11,000, 12,000, 13,000, or 13,659 nucleotides of SEQ ID NO: 14. In certain
embodiments,
such allelic variants of INHT26 DNA molecules include sequences having at
least 85%, 90%,
95%, 98%, or 99% sequence identity across the entire length of SEQ ID NO: 14,
16, or 17.
[0061] Also provided are unique transgenic locus excision sites created by
excision of INHT26
transgenic loci or selectively excisable INHT26 transgenic loci, DNA molecules
comprising
the INHT26 transgenic loci or unique fragments thereof (i.e., fragments of an
INHT26 locus
which are not found in an DA544406-6 transgenic locus), INHT26 plants
comprising the same,
biological samples containing the DNA, nucleic acid markers adapted for
detecting the DNA
molecules, and related methods of identifying soybean plants comprising unique
INHT26
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transgenic locus excision sites and unique fragments of a INHT26 transgenic
locus. An
example of such an excision site would include an excision site created by
excising the INHT26
transgenic locus with a guide RNA encoded by SEQ ID NO:19 and a suitable Cas
RdDe (e.g.,
a Cas12a nuclease of SEQ ID NO: 15). DNA molecules comprising unique fragments
of an
INHT26 transgenic locus are diagnostic for the presence of an INHT26
transgenic locus or
fragments thereof in a soybean plant, soybean cell, soybean seed, products
obtained therefrom
(e.g., seed meal or stover), and biological samples. DNA molecules comprising
unique
fragments of an INHT26 transgenic locus include DNA molecules comprising the
CgRRS
include SEQ ID NO: 17.
[0062] 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 soybean DA544406-6 transgenic locus. The soybean DA544406-6 transgenic
locus and
its transgenic junction sequences are also depicted in Figure 1. Soybean
plants comprising the
DA544406-6 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 DA544406-6 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
"genbitgroup.com/en/gmo/gmodatabase"), and the Biosafety Clearing-House (BCH)
database
(available on the http internet site "bch.cbd.int/database/organisms").
[0063] Sequences of the junction polynucleotides as well as the transgenic
insert(s) of the
DA544406-6 transgenic locus which can be improved by the methods provided
herein are set
forth or otherwise provided in SEQ ID NO: 1, US 9,540,655, the sequence of the
DA544406-
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6 locus in the deposited seed of ATCC accession No. PTA-11336, and elsewhere
in this
disclosure. In certain embodiments provided herein, the DAS44406-6 transgenic
locus set forth
in SEQ ID NO: 1 or present in the deposited seed of ATCC accession No. PTA-
11336 is
referred to as an "original DA544406-6 transgenic locus." Allelic or other
variants of the
sequence set forth SEQ ID NO: 1, 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 DA544406-6 transgenic plant loci (e.g., progeny of deposited seed of
accession No.
PTA-11336 which contain allelic variants of SEQ ID NO:1 or progeny originating
from
transgenic plant cells comprising the original MIR162 transgenic set forth in
US 9,540,655)
can also be improved by identifying sequences in the variants that correspond
to the SEQ ID
NO: 1 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, 11,000, 12,000, 13,000 or 13659 nucleotides of SEQ ID NO: 1.
Also provided
are plants, plant parts including seeds, genomic DNA, and/or DNA obtained from
INHT26
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 INHT26
transgenic locus
or a portion thereof. Also provided herein are methods of detecting plants,
genomic DNA,
and/or DNA obtained from plants comprising a INHT26 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
DA544406-6
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, 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, 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, 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, allelic variants thereof, or other variants thereof to obtain an INHT26
transgenic locus. In
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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,
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 DA544406-6 transgenic locus of SEQ ID NO: 1, allelic
variants thereof,
or other variants thereof to obtain an INHT26 transgenic locus. In other
embodiments, the
OgRRS is found in non-transgenic DNA or transgenic DNA of the 3' junction
polynucleotide
of the DA544406-6 transgenic locus of any one of SEQ ID NO: 1, 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 polynucleotide
of the
transgenic locus of SEQ ID NO: 1, allelic variants thereof, or other variants
thereof to obtain
an INHT26 transgenic locus.
[0064] 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 INHT26 transgenic locus, in transgenic DNA of a DNA
junction
polynucleotide of an INHT26 transgenic locus or can span the junction of the
transgenic and
non-transgenic DNA of a DNA junction polynucleotide of an INHT26 transgenic
locus. An
OgRRS can likewise be located in non-transgenic plant genomic DNA of a DNA
junction
polynucleotide of an INHT26 transgenic locus, in transgenic DNA of a DNA
junction
polynucleotide of an INHT26 transgenic locus, or can span the junction of the
transgenic and
non-transgenic DNA of a DNA junction polynucleotide of an INHT26 transgenic
locus
[0065] 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
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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 embodiments, at least two transgenic
loci (e.g., transgenic
loci including an INHT26 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 3 (bottom
"Alternative" panel), where two or more of the transgenic loci ("Event" in
Figure 3) are
provided in Line A and then moved into elite crop plant germplasm by
introgression. In the
non-limiting Figure 3 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 3)
comprising two or
more of the modified transgenic loci. This elite germplasm containing the
modified transgenic
loci (e.g., "Universal Donor" of Figure 3) can then be subjected to genome
editing molecules
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which can excise at least one of the transgenic loci ("Event Removal" in
Figure 3) and introduce
other targeted genetic changes ("GE" in Figure 3) 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 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 3). 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 soybean plants comprising
the INHT26
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.
[0066] 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
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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.
[0067] 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 INHT26 transgenic locus
comprising an
OgRRS in non-transgenic DNA of a 1" junction polynucleotide sequence and a
CgRRS in a
2nd junction polynucleotide sequence is deleted with a gRNA and RdDe that
recognize the
OgRRS and the CgRRS to produce an INHT26 transgenic locus excision site. For
example, an
INHT26 transgenic locus set forth in SEQ ID NO: 14 can be deleted with a
Cas12a RdDe (e.g.
the Cas12a of SEQ ID NO: 15) and a gRNA comprising an RNA encoded by SEQ ID
NO: 19.
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
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
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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 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 INHT26 transgenic loci excision sites
are provided
herein.
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[0068] In other embodiments, a segment comprising a INHT26 transgenic locus
(e.g., a
transgenic locus comprising an OgRRS in non-transgenic DNA of a 1st 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 (e.g., the Cas12a RdDe of SEQ ID NO: 15 and a gRNA
comprising
an RNA encoded by SEQ ID NO: 19) 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 4C, 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 100 nucleotides
are changed relative to the endogenous DNA at the essentially restored
excision site.
[0069] 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 1st and a 2nd 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 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
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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 DA544406-6 transgenic locus.
[0070] 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 originator gRNA Recognition Site (OgRRS) to provide for cleavage
within the
junction polynucleotides which flank an INHT26 transgenic locus. In certain
embodiments,
the CgRRS/gRNA and OgRRS/gRNA hybridization complexes are recognized by the
same
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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/sku402). In certain
embodiments, an
operably linked PAM site is co-introduced with the gRNA hybridization site
polynucleotide at
the CgRRS. In certain embodiments, the gRNA hybridization site polynucleotides
are
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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 soybean 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 used to
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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 is created in a DNA
sequence are
illustrated in Example 2 and Figure 2.
[0071] 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 (e.g., the Cas12a RdDe of SEQ ID NO: 15 and a gRNA comprising
an
RNA encoded by SEQ ID NO: 19). 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 4B. In the depicted
example set
forth in Figure 4B, 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 partially
or essentially restored.
[0072] In certain embodiments, edited transgenic plant genomes provided herein
can lack one
or more selectable markers found in an original event (transgenic locus).
Original DA544406-
6 transgenic loci (events), including those set forth in SEQ ID NO: 1), US
9,540,655, the
sequence of the DA544406-6 locus in the deposited seed of accession No. PTA-
11336 and
progeny thereof, contain a selectable marker gene encoding a phosphinotricin
acetyl transferase
(PAT) protein which confers tolerance to the herbicide glufosinate. In certain
embodiments
provided herein, the DNA element comprising, consisting essentially of, or
consisting of the
PAT selectable marker gene of an DA544406-6 transgenic locus is absent from an
INHT26
transgenic locus. The PAT selectable marker cassette can be excised from an
original
DA544406-6 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 PAT 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 PAT selectable marker transgene) and selecting for plant cells,
plant parts, or
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plants wherein the selectable marker has been excised. In certain embodiments,
the selectable
or scoreable marker transgene can be inactivated. 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 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 PAT 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 PAT selectable marker
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 PAT
selectable 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 PAT
selectable marker transgene.
[0073] 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 4C, the donor DNA template can comprise an
expression
cassette flanked by DNA homologous to non-transgenic DNA located telomere
proximal and
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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 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, 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.
[0074] In certain embodiments, INHT26 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 3,
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,
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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
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; (I) 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]plishare/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
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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 soybean genes that can be
subjected to
targeted gene edits to confer useful traits include: (a) ZmIPK1 (herbicide
tolerant and phytate
reduced soybean; Shukla et al., Nature. 2009;459:437-41); (b) ZmGL2 (reduced
epicuticular
wax in leaves; Char et al. Plant Biotechnol J. 2015;13:1002); (c) ZmMTL
(induction of haploid
plants; Kelliher et al. Nature. 2017;542:105); (d) Wxl (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 soybean 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).
[0075] 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 (ZFN) 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
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thereof) suitable for insertion at a break in genomic DNA (e.g., by non-
homologous end joining
(NHEJ) or microhomology-mediated end joining (MMEJ).
[0076] 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 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) used to form an RNA-guided endonuclease/guide RNA
complex can
specifically bind via hybridization to gRNA hybridization site sequences
(i.e., protospacer
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
(e.g., the Cas12a
protein of SEQ ID NO: 15). 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).
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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 to a
recurrent parent).
Multiple endonucleases can be provided in expression cassettes with the
appropriate promoters
to allow multiple genome site editing.
[0077] CRISPR technology for editing the genes of eukaryotes is disclosed in
US Patent
Application Publications 2016/0138008A1 and U52015/0344912A1, 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
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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.
[0078] 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. 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-
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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 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.
[0079] 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
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Cas9 DlOA 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.
[0080] 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 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
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(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
and can be
adapted for use in the methods described herein; see, e.g., Guo et at. (2010)
1 Mol. Biol.,
400:96 - 107.
[0081] 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
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methods described herein (Wu et al.; Biochem Biophys Res Commun.
(2014);446(1):261-6;
Luo eta!; Scientific Reports 6, Article number: 20657 (2016)).
[0082] 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 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
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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, 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
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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 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
polynucleotide and include the donor DNA template set forth in SEQ ID NO: 11
and
equivalents thereof with longer or shorter homology arms. In certain
embodiments, a donor
DNA template can comprise an adapter molecule (e.g., a donor DNA template
formed by
annealing single stranded DNAs which do not overlap at their 5' and 3'
terminal ends) with
cohesive ends which can anneal to an overhanging cleavage site (e.g.,
introduced by a Cas12a
nuclease and suitable gRNAs). 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.
[0083] 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
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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 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
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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 polynucleotide. An example of a useful DNA donor template
provided
herein is a DNA molecule comprising SEQ ID NO: 11.
[0084] Various treatments are useful in delivery of gene editing molecules
and/or other
molecules to a DA544406-6 or INHT26 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 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
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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.
[0085] 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 DA544406-6 or
INHT26 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 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,
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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 soybean, 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 soybean chloroplast aldolase promoter as disclosed in US
Patent 7,151,204,
and the nopaline synthase (NOS) and octopine synthase (OCS) 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
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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.
[0086] 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.
[0087] In certain embodiments, the DA544406-6 or INHT26 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 the centromere-specific histone CENH3, as described
by
Maruthachalam and Chan in "How to make haploid Arabidopsis thaliana", protocol
available
at www [dot] op enwetware [dot] org/im age s/d/d3/Hapl oi d Arab i dop si
s_protocol [dot]p df; (Ravi
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et al. (2014) Nature Communications, 5:5334, doi: 10.1038/nc0mms6334).
Haploids can also
be obtained in a wide variety of monocot plants (e.g., soybean, 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 soybean lines that can be used to
obtain haploid
soybean 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.
[0088] In certain embodiments, the DA544406-6 or INHT26 plant cells used in
the methods
provided herein can include non-dividing cells. Such non-dividing cells can
include plant cell
protoplasts, plant cells subjected to one or more of a genetic and/or
pharmaceutically-induced
cell-cycle blockage, and the like.
[0089] In certain embodiments, the DA544406-6 or INHT26 plant cells in used in
the methods
provided herein can include dividing cells. Dividing cells can include those
cells found in
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various plant tissues including leaves, meristems, and embryos. These tissues
include dividing
cells from young soybean leaf, meristems and scutellar tissue from about 8 or
10 to about 12
or 14 days after pollination (DAP) embryos. The isolation of soybean 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 soybean 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.
[0090] In some embodiments, methods provided herein can include the additional
step of
growing or regenerating an INHT26 plant from a INHT26 plant cell that had been
subjected to
the gene editing or from a regenerable plant structure obtained from that
INHT26 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
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
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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
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
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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
INHT26
plant or its seeds, including: (a) soybean 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 soybean 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.
EMBODIMENTS
[0091] Various embodiments of the plants, genomes, methods, biological
samples, and other
compositions described herein are set forth in the following sets of numbered
embodiments.
[0092] la. A transgenic soybean plant cell comprising an INHT26
transgenic locus
comprising an originator guide RNA recognition site (OgRRS) in a first DNA
junction
polynucleotide of a DAS44406-6 transgenic locus and a cognate guide RNA
recognition site
(CgRRS) in a second DNA junction polynucleotide of the DAS44406-6 transgenic
locus.
[0093] lb. A transgenic soybean plant cell comprising an INHT26
transgenic locus
comprising an insertion and/or substitution of DNA in a DNA junction
polynucleotide of a
DAS44406-6 transgenic locus with DNA comprising a cognate guide RNA
recognition site
(CgRRS).
[0094] 2. The transgenic soybean plant cell of embodiment la or lb,
wherein said
CgRRS comprises the DNA molecule set forth in SEQ ID NO: 16 or 17; and/or
wherein said
DA544406-6 transgenic locus is set forth in SEQ ID NO:1, is present in seed
deposited at the
ATCC under accession No. PTA-11336, is present in progeny thereof, is present
in allelic
variants thereof, or is present in other variants thereof.
[0095] 3. The transgenic soybean plant cell of embodiments la, lb, or 2,
wherein said
INHT26 transgenic locus comprises the DNA molecule set forth in SEQ ID NO: 14
or an allelic
variant thereof.
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[0096] 4. A transgenic soybean plant part comprising the soybean plant
cell of any one
of embodiments la, lb, 2, or 3, wherein said soybean plant part is optionally
a seed.
[0097] 5. A transgenic soybean plant comprising the soybean plant cell of
any one of
embodiments la, lb, 2, or 3.
[0098] 6. A method for obtaining a bulked population of inbred seed
comprising selfing
the transgenic soybean plant of embodiment 5 and harvesting seed comprising
the INHT26
transgenic locus from the selfed soybean plant.
[0099] 7. A method of obtaining hybrid soybean seed comprising crossing
the
transgenic soybean plant of embodiment 5 to a second soybean plant which is
genetically
distinct from the first soybean plant and harvesting seed comprising the
INHT26 transgenic
locus from the cross.
[00100] 8. A DNA molecule comprising SEQ ID NO: 14, 16, 17, 14 or an
allelic variant
thereof
[00101] 9. A processed transgenic soybean plant product comprising the DNA
molecule
of embodiment 8.
[00102] 10. A biological sample containing the DNA molecule of embodiment
8.
[00103] 11. A nucleic acid molecule adapted for detection of genomic DNA
comprising
the DNA molecule of embodiment 8, wherein said nucleic acid molecule
optionally comprises
a detectable label.
[00104] 12. A method of detecting a soybean plant cell comprising the
INHT26
transgenic locus of any one of embodiments la, lb, 2, or 3, comprising the
step of detecting
DNA molecule comprising SEQ ID NO: 14, 16, 17, 14 or an allelic variant
thereof.
[00105] 13. A method of excising the INHT26 transgenic locus from the
genome of the
soybean plant cell of any one of embodiments la, lb, 2, or 3, comprising the
steps of:
(a) contacting the plant cell or genome thereof 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 INHT26 transgenic locus flanked by the OgRRS and the CgRRS has been
excised.
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Examples
[00106] Example 1. Application of a Cas12a RNA guided endonuclease and
guide
RNAs to change or excise the 3' -T-DNA junction sequence in the DAS44406 event
[00107] The DAS44406 3' junction polynucleotide sequence set forth in SEQ
ID NO: 3
is flanked by five Cas12a recognition sequences. The Guide-1 and Guide-2
sequences are
located at 5' -end of SEQ ID NO: 3 and Guides-3-5 lie within the 3' junction
polynucleotide
sequence of SEQ ID NO: 3. These can be used to modify some of the 3' junction
polynucleotide
sequence or eliminate most of it. There are several iterations of this
approach. In one
embodiment, Guide-3, Guide-4, or Guide-5 are used alone to disrupt the
DA544406 3'-junction
sequence (e.g., by using a Cas12a endonuclease and 1 of Guide-3, Guide-4, or
Guide-5 to
cleave the 3' junction polynucleotide sequence and recovering genomic edits
where the 3'
DNA junction polynucleotide sequence of DA544406 is disrupted. In another
embodiment,
Guide-1 or Guide-2 is used with either Guide-3, Guide-4 or Guide-5 to
eliminate most of the
DA544406 3' junction polynucleotide sequence.
[00108] Table One. Description of Guide RNAs and SEQ ID NO of DNA encoding
same
SEQ ID Start-End in SEQ ID Strand of
Guide RNA ID NO NO: 1 SEQ ID NO: 1 PAM
4
Guide-1 11656-11678 -1 TTTA
Guide-2 11646-11688 1 TTTA
Guide-3 6 11725-11747 1 TTTG
7
Guide-4 11730-11752 1 TTTA
Guide-5 8 11773-11795 1 TTTA
[00109] The Cas12a nuclease and the single or combined guide RNAs are
introduced
into soybean plant cells containing the DA544406-6 event. In certain
embodiments, the Cas12a
nuclease and gRNA(s) are encoded and expressed from a T-DNA transformed into
the
DA544406-6 event via Agrobacterium-mediated transformation. Alternatively, the
T-DNA
can be transformed into any convenient soy line, and then crossed with the
DA544406-6 event
to combine the Cas12a ribonucleoprotein expressing T-DNA with the DA544406-6
event. The
Cas12a nuclease and gRNAs can also be assembled in vitro then delivered to
DA544406-6
explants as ribonucleoprotein complexes using a biolistic approach (Svitashev
et al., Nat
Commun. 2016; 7:13274; Zhang et al., 2021, Plant Commun. 2(2):100168). Also, a
plasmid
encoding a Cas12a nuclease and the gRNA(s) can be delivered to DA544406-6
explants using
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a biolistic approach. This will produce plant cells that have a high
likelihood of incurring
mutations that disrupt the DAS44406-6 3' junction polynucleotide sequence.
[00110] In the Agrobacterium approach, a binary vector that contains a
strong
constitutive expression cassette like the AtUbi10 promoter: :AtUbi10
terminator driving
Cas12a, a PolII or PolII gene cassette driving the Cas12a gRNA(s) and a CaMV
35S:NPTII:NOS or other suitable plant selectable marker (e.g., a
phosphomannose isomerase
(Reed et al. 2001, In Vitro Cellular & Developmental Biology - Plant 37: 127-
132) or
hygromycin phosphotransferase (Itaya, et al. 2018, In Vitro Cellular &
Developmental Biology
-Plant 54: 184-194)) is constructed and cells comprising the integrated T-
DNA(s) are selected
using an appropriate selection agent. An expression cassette driving a
fluorescent protein like
mScarlet may also be useful to monitor the plant transformation process.
[00111] The T-DNA-based expression cassettes are delivered from
superbinary vectors
in Agrobacterium strain LBA4404. Soy transformations are performed based on
published
methods (Zhang et al., 1999, Plant Cell, Tissue and Organ Culture 56(1), 37-
46). Briefly,
cotyledonary explants are prepared from the 5-day-old soybean seedlings by
making a
horizontal slice through the hypocotyl region, approximately 3-5 mm below the
cotyledon. A
subsequent vertical slice is made between the cotyledons, and the embryonic
axis is removed.
This generates 2 cotyledonary node explants. Approximately 7-12 vertical
slices are made on
the adaxial surface of the explant about the area encompassing 3 mm above the
cotyledon/hypocotyl junction and 1 mm below the cotyledon/hypocotyl junction.
Explant
manipulations are done with a No. 15 scalpel blade.
[00112] Explants are immersed in the Agrobacterium inoculum for 30 min and
then co-
cultured on 100 x 15 mm Petri plates containing the Agrobacterium resuspension
medium
solidified with 0.5% purified agar (BBL Cat # 11853). The co-cultivation
plates are overlaid
with a piece of Whatman #1 filter paper (Mullins et al., 1990; Janssen and
Gardner, 1993;
Zhang et al., 1997). The explants (5 per plate) are cultured adaxial side down
on the co-
cultivation plates, that are overlaid with filter paper, for 3 days at 24 C,
under an 18/6 hour
light regime with an approximate light intensity of 80 Ilmol s-1 m-2
(F17T8/750 cool white
bulbs, Litetronicsc)). The co-cultivation plates are wrapped with Parafilm .
Following the co-cultivation period explants are briefly washed in B5 medium
supplemented
with 1.67 mg 1-1 BAP, 3% sucrose, 500 mg 1-1 ticarcillin and 100 mg 1-1
cefotaxime. The
medium is buffered with 3 mM MES, pH 5.6. Growth regulator, vitamins and
antibiotics are
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filter sterilized post autoclaving. Following the washing step, explants are
cultured (5 per plate)
in 100 x 20 mm Petri plates, adaxial side up with the hypocotyl imbedded in
the medium,
containing the washing medium solidified with 0.8% purified agar (BBL Cat #
11853)
amended with either G418, neomycin, or kanamycin at concentrations permitting
selection of
transformants. This medium is referred to as shoot initiation medium (SI).
Plates are wrapped
with 3M pressure sensitive tape (Scotch, 3M, USA) and cultured under the
environmental
conditions used during the seed germination step (at 24 C, 18/6 light regime,
under a light
intensity of approximately 150 [tmol s m' .
[00113] After 2 weeks of culture, the hypocotyl region is excised from
each of the
explants, and the remaining explant, cotyledon with differentiating node, is
subsequently
subcultured onto fresh SI medium. Following an additional 2 weeks of culture
on SI medium,
the cotyledons are removed from the differentiating node. The differentiating
node is
subcultured to shoot elongation medium (SE) composed of Murashige and Skoog
(MS) (1962)
basal salts, B5 vitamins, 1 mg 1-1 zeatin-riboside, 0.5 mg 1-1 GA3 and 0.1 mg
1-1 IAA, 50 mg 1-
1 glutamine, 50 mg 1-1 asparagine, 3% sucrose and 3 mM MES, pH 5.6. The SE
medium is
amended with G418, neomycin, or kanamycin at concentrations permitting
selection of
transformants. The explants are subcultured biweekly to fresh SI medium until
shoots reach a
length greater than 3 cm. The elongated shoots are rooted on Murashige and
Skoog salts with
B5 vitamins, 1% sucrose, 0.5 mg 11 NAA without further selection in Magenta
boxes .
[00114] When a sufficient amount of viable tissue is obtained, it can be
screened for
mutations at the DA544406-6 junction sequence, using a PCR-based approach. One
way to
screen is to design DNA oligonucleotide primers that flank and amplify the
DA544406-6
junction plus surrounding sequence. For example, the primers (5'-
AGCGGCCGGGTTTCTAGTCACCGGT-3'; SEQ ID NO: 9) and (5'-
TCTCATTTTCACACATATACATGCA-3'; SEQ ID NO: 10) will produce a ¨440 bp product
in a PCR reaction 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 DA544406-6 3'-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.
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[00115] Example 2. Insertion of a CgRRS element in the 3'-junction of the
DAS44406-
6 event.
[00116] 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 their entireties. DNA sequences encoding
the c2 NLS-Exo,
c2 NLS lambda beta SSAP, and c2NLS-SSB fusion proteins are operably linked to
suitable
promoter(s) (e.g., AtUbil0, CaMV35S, and/or SlUbil0 promoter) and suitable
polyadenylation
site(s) (e.g., nos 3', PeaE9 3', tmr 3', tms 3', AtUbi 10 3', and tr7 3'
elements), to provide the
exonuclease, S SAP, and SSB plant expression cassettes.
[00117] A DNA donor template sequence (SEQ ID NO: 11) that targets the 3'-
T-DNA
junction polynucleotide of the DA544406-6 event (SEQ ID NO:1; Figure 1) for
HDR-mediated
insertion of a 27 base pair OgRRS sequence (SEQ ID NO: 18) that is identical
to a Cas12a
recognition site at the 5'-junction polynucleotide of the DA544406-6 T-DNA
insert is
constructed. The DNA donor sequence includes a replacement template with
desired insertion
region (27 base pairs long) 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 genomic DNA insertion site (SEQ ID NO: 3) in the DA544406-6
transgenic locus
(SEQ ID NO: 1). The replacement template region comprising the donor DNA is
flanked at
each end by DNA sequences identical to the DA544406-6 3' junction
polynucleotide sequence
and contains a CgRRS element recognized by the same Cas12a RNA-guided nuclease
and a
gRNA (e.g., comprising an RNA encoded by SEQ ID NO: 19) that recognize the
OgRRS
located in the 5' junction polynucleotide.
[00118] 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 (e.g., encoded by SEQ ID NO: 8)
complementary to
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sequences adjacent to the insertion site is constructed. An Agrobacterium
superbinary plasmid
transformation vector containing a cassette that provides for the expression
of a suitable plant
selectable marker (e.g., a neomycin phosphotransferase (nptII) or hygromycin
phosphotransferase (hpt)) is constructed. Once the cassettes, donor sequence
and
Agrobacterium superbinary plasmid transformation vector are constructed, they
are combined
to generate two soybean transformation plasmids. In other embodiments, other
gRNAs (Guide-
3 or Guide-4 alone or Guide -1 or Guide-2 with either Guide-3, Guide-4 or
Guide-5) can be
used to introduce double stranded breaks in the DAS44406-6 3' junction
polynucleotide for
insertion of a CgRRS using similar donor DNA templates and the aforementioned
Cas12a,
SSAP, SSB, and EXO reagents.
[00119] A soybean transformation plasmid is constructed with a neomycin
phosphotransferase (nptII) or hygromycin phosphotransferase (hpt) cassette,
the RNA-guided
sequence-specific endonuclease cassette, the guide RNA cassette, and the
DAS44406-6 3'-
T DNA junction sequence DNA donor sequence into the Agrobacterium superbinary
plasmid
transformation vector (the control vector).
[00120] A soybean transformation plasmid is constructed with a neomycin
phosphotransferase (nptII) or hygromycin phosphotransferase (hpt) 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 DAS44406-63'-T DNA junction
sequence donor
DNA template sequence (SEQ ID NO: 11) into the Agrobacterium superbinary
plasmid
transformation vector (the lambda red vector).
[00121] All constructs are transformed into Agrobacterium strain LBA4404.
[00122] Soybean 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
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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 G418 or hygromycin at concentrations permitting
selection of
transformants when a nptII or hpt selectable marker, respectively, is used 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 mg/L PPT to initiate shoot development.
Calli remains on
Pre-Regeneration media for 7 days. Calli beginning to initiate shoots are
transferred to
Regeneration medium with G418 or hygromycin at concentrations permitting
selection of
transformants when a nptII or hpt selectable marker is used 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.
[00123] When a sufficient amount of viable tissue is obtained, it can be
screened for
insertion at the DA544406-6 junction sequence, using a PCR-based approach. The
PCR primer
on the 5'-end is 5'-TATTGTCGCCGTATGTAATCGGCGT-3' (SEQ ID NO: 12). The PCR
primer on the 3'-end is 5'-TTTTAGTTCAAGTCAACTTGTCAGT--3' (SEQ ID NO: 13). The
above primers that flank donor DNA homology arms are used to amplify the
DA544406-6 3'-
junction polynucleotide sequence. The correct donor sequence insertion will
produce a 1366
bp product. A unique DNA fragment comprising the CgRRS in the DA544406-6 3'
junction
polynucleotide is set forth in SEQ ID NO: 17. 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 DA544406-6 junction sequence now
contains the
intended Cas12a recognition sequence 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 INHT26 transgenic locus (SEQ ID NO: 14)
comprising
the CgRRS and OgRRS (e.g., which each comprise SEQ ID NO: 18) can be excised
using
Cas12a and a suitable gRNA which hybridizes to DNA comprising SEQ ID NO: 19 at
both the
OgRRS and the CgRRS.
[00124] The breadth and scope of the present disclosure should not be
limited by any of
the above-described embodiments.
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