Note: Descriptions are shown in the official language in which they were submitted.
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INIR19 TRANSGENIC SOYBEAN
REFERENCE TO SEQUENCE LISTING SUBMITTED
ELECTRONICALLY
100011 The sequence listing contained in the file named "10104W01 ST25.txt,"
which was
created on July 29, 2021 and electronically filed on July 30, 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
examples of a selected transgenic soybean event which confers lepidopteran
insect pest tolerance
and herbicide tolerance is the DA581419 transgenic soybean event disclosed in
U.S. Patent No.
8,680,363. DA581419 transgenic soybean plants express a Cry 1F and Cry lAc
proteins which
confer tolerance to lepidopteran insect pests that include Pseudoplusia
includens (soybean looper),
Anticarsia gemmatalis (velvet bean caterpillar) and Spodoptera frugiperda
(fall armyworm).
DA581419 transgenic soybean plants express a PAT (phosphinothricin
acetyltransferase) which
confers tolerance to the herbicide phosphinothricin (glufosinate).
[0004] Methods for removing selectable marker genes and/or duplicated
transgenes in transgene
insertion sites in plant genomes involving use of site-specific recombinase
systems (e.g., cre-lox)
as well as for insertion of new genes into transgene insertion sites have been
disclosed (Srivastava
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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 INIR19 transgenic
locus comprising
an originator guide RNA recognition site (OgRRS) in a first DNA junction
polynucleotide of a
DAS81419 transgenic locus and a cognate guide RNA recognition site (CgRRS) in
a second DNA
junction polynucleotide of the DAS81419 transgenic locus are provided.
Transgenic soybean plant
cells comprising an INIR19 transgenic locus comprising an insertion and/or
substitution in a DNA
junction polynucleotide of a DAS81419 transgenic locus of DNA comprising a
cognate guide RNA
recognition site (CgRRS) are provided. In certain embodiments, the DA581419
transgenic locus
is set forth in SEQ ID NO:1, is present in seed deposited at the ATCC under
accession No. PTA-
12006, is present in progeny thereof, is present in allelic variants thereof,
or is present in other
variants thereof INIR19 transgenic soybean plant cells, transgenic soybean
plant seeds, and
transgenic soybean plants all comprising a transgenic locus set forth in SEQ
ID NO: 2, 3, 17, and
allelic variants thereof 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
INIR19 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 INIR19 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: 2, 3, 17, or an allelic variant thereof
and harvesting
transgenic seed comprising the transgenic locus set forth in SEQ ID NO: 2, 3,
17, or allelic variant
thereof are provided.
[0008] A DNA molecule comprising SEQ ID NO: 2, 3, 8, 9, 10, 11, 17, 23,
or an allelic
variant thereof is provided. Processed transgenic soybean plant products and
biological samples
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comprising the DNA molecules are provided. Nucleic acid molecules adapted for
detection of
genomic DNA comprising the DNA molecules, wherein said nucleic acid molecule
optionally
comprises a detectable label are provided. Methods of detecting a soybean
plant cell comprising
an INIR19 transgenic locus, comprising the step of detecting a DNA molecule
comprising SEQ ID
NO: 2,3, 8,9, 10, 11, 17, 23, or an allelic variant thereof are provided.
[0009]
Methods of excising the INIR19 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 INIR19 transgenic locus flanked by the OgRRS and the CgRRS has been
excised.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
100101
The patent or application file contains at least one drawing executed in
color.
Copies of this patent or patent application publication with color drawing(s)
will be provided by
the Office upon request and payment of the necessary fee.
[0011]
Figure 1 shows a schematic diagram which compares current breeding strategies
for introgression of transgenic events (i.e., transgenic loci) to alternative
breeding strategies for
introgression of transgenic events where the transgenic events (i.e.,
transgenic loci) can be removed
following introgression to provide different combinations of transgenic
traits. In Figure 2, "GE"
refers to genome editing (e.g., including introduction of targeted genetic
changes with genome
editing molecules and "Event Removal" refers to excision of a transgenic locus
(i.e., an "Event")
with genome editing molecules.
[0012]
Figure 2A, B, C. Figure 2A 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 2B 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
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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 2C 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 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
2C, 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 1" 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
[0013] 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.
[0014] Where a term is provided in the singular, the inventors also
contemplate
embodiments described by the plural of that term.
[0015] 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.
[0016] 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.
[0017] 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),
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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).
[0018] As used herein, the phrase "approved transgenic locus" is a
genetically modified
plant event which has been authorized, approved, and/or de-regulated for any
one of field testing,
cultivation, human consumption, animal consumption, and/or import by a
governmental body.
Illustrative and non-limiting examples of governmental bodies which provide
such approvals
include the Ministry of Agriculture of Argentina, Food Standards Australia New
Zealand, National
Biosafety Technical Committee (CTNBio) of Brazil, Canadian Food Inspection
Agency, China
Ministry of Agriculture Biosafety Network, European Food Safety Authority, US
Department of
Agriculture, US Department of Environmental Protection, and US Food and Drug
Administration.
[0019] The term "backcross", as used herein, refers to crossing an Fl
plant or plants with
one of the original parents. A backcross is used to maintain or establish the
identity of one parent
(species) and to incorporate a particular trait from a second parent
(species). The term "backcross
generation", as used herein, refers to the offspring of a backcross.
[0020] 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.
[0021] 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).
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[0022] 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: 18.
[0023] The term "crossing" as used herein refers to the fertilization of
female plants (or
gametes) by male plants (or gametes). The term "gamete" refers to the haploid
reproductive cell
(egg or pollen) produced in plants by meiosis from a gametophyte and involved
in sexual
reproduction, during which two gametes of opposite sex fuse to form a diploid
zygote. The term
generally includes reference to a pollen (including the sperm cell) and an
ovule (including the
ovum). When referring to crossing in the context of achieving the
introgression of a genomic region
or segment, the skilled person will understand that in order to achieve the
introgression of only a
part of a chromosome of one plant into the chromosome of another plant, random
portions of the
genomes of both parental lines recombine during the cross due to the
occurrence of crossing-over
events in the production of the gametes in the parent lines. Therefore, the
genomes of both parents
must be combined in a single cell by a cross, where after the production of
gametes from the cell
and their fusion in fertilization will result in an introgression event.
[0024] 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
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determined by comparing non-transgenic genomic sequence of a sequenced non-
transgenic plant
genome to the non-transgenic DNA in the junction polynucleotides.
[0025] 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.
[0026] As used herein, the term "DAS81419" 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.
8,680,363, which is incorporated herein by reference in its entirety.
Representative DAS81419
transgenic soybean seed have been deposited with American Type Culture
Collection (ATCC,
Manassas, Va. 20110-2209 USA) under Accession No. PTA-12006. DA581419
transgenic loci
include loci having the sequence of SEQ ID NO:1, the sequence of the DA581419
locus in the
deposited seed of Accession No. PTA-12006 and any progeny thereof, as well as
allelic variants
and other variants of SEQ ID NO: 1.
[0027] 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.
[0028] As used herein, the phrase "elite crop plant" refers to a plant
which has undergone
breeding to provide one or more trait improvements. Elite crop plant lines
include plants which
are an essentially homozygous, e.g., inbred or doubled haploid. Elite crop
plants can include inbred
lines used as is or used as pollen donors or pollen recipients in hybrid seed
production (e.g., used
to produce Fl plants). Elite crop plants can include inbred lines which are
selfed to produce non-
hybrid cultivars or varieties or to produce (e.g., bulk up) pollen donor or
recipient lines for hybrid
seed production. Elite crop plants can include hybrid Fl progeny of a cross
between two distinct
elite inbred or doubled haploid plant lines.
[0029] As used herein, an "event," "a transgenic event," "a transgenic
locus" and related
phrases refer to an insertion of one or more transgenes at a unique site in
the genome of a plant as
well as to DNA fragments, plant cells, plants, and plant parts (e.g., seeds)
comprising genomic
DNA containing the transgene insertion. Such events typically comprise both a
5' and a 3' DNA
junction polynucleotide and confer one or more useful traits including
herbicide tolerance, insect
resistance, male sterility, and the like.
[0030] As used herein, the phrases "endogenous sequence," "endogenous
gene,"
"endogenous DNA," "endogenous polynucleotide," and the like refer to the
native form of a
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polynucleotide, gene or polypeptide in its natural location in the organism or
in the genome of an
organism.
[0031] 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.
[0032] As used herein, the term "F1" refers to any offspring of a cross
between two
genetically unlike individuals.
[0033] 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.
[0034] 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.
[0035] As used herein, the term "INIR19" is used to refer either
individually collectively
to items that include any or all of the DAS81419 transgenic soybean loci which
have been modified
as disclosed herein, modified DAS81419 transgenic soybean plants and parts
thereof including
seed, and DNA obtained therefrom.
[0036] The term "isolated" as used herein means having been removed from
its natural
environment.
[0037] As used herein, the terms "include," "includes," and "including"
are to be construed
as at least having the features to which they refer while not excluding any
additional unspecified
features.
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[0038] As used herein, the phrase "introduced transgene" is a transgene
not present in the
original transgenic locus in the genome of an initial transgenic event or in
the genome of a progeny
line obtained from the initial transgenic event. Examples of introduced
transgenes include
exogenous transgenes which are inserted in a resident original transgenic
locus.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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).
[0043] The term "offspring", as used herein, refers to any progeny
generation resulting
from crossing, selfing, or other propagation technique.
[0044] 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,
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a promoter is operably linked to a coding sequence if the promoter affects its
transcription or
expression. When the phrase "operably linked" is used in the context of a PAM
site and a guide
RNA hybridization site, it refers to a PAM site which permits cleavage of at
least one strand of
DNA in a polynucleotide with an RNA dependent DNA endonuclease or RNA
dependent DNA
nickase which recognize the PAM site when a guide RNA complementary to guide
RNA
hybridization site sequences adjacent to the PAM site is present. A OgRRS and
its CgRRS 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.
[0045] 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.
[0046] 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.
[0047] 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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[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] As used herein, the phrase "originator guide RNA recognition site"
or the acronym
"OgRRS" refers to an endogenous DNA polynucleotide comprising a protospacer
adjacent motif
(PAM) site operably linked to a guide RNA hybridization site. In certain
embodiments, an OgRRS
can be located in an untransformed plant chromosome or in non-transgenic DNA
of a DNA
junction polynucleotide of both an original transgenic locus and a modified
transgenic locus. In
certain embodiments, an OgRRS can be located in transgenic DNA of a DNA
junction
polynucleotide of both an original transgenic locus and a modified transgenic
locus. In certain
embodiments, an OgRRS can be located in both transgenic DNA and non-transgenic
DNA of a
DNA junction polynucleotide of both an original transgenic locus and a
modified transgenic locus
(i.e., can span transgenic and non-transgenic DNA in a DNA junction
polynucleotide).
[0053] As used herein the phrase "cognate guide RNA recognition site" or
the acronym
"CgRRS" refer to a DNA polynucleotide comprising a PAM site operably linked to
a guide RNA
hybridization site, where the CgRRS is absent from transgenic plant genomes
comprising a first
original transgenic locus that is unmodified and where the CgRRS and its
corresponding OgRRS
can hybridize to a single gRNA. A CgRRS can be located in transgenic DNA of a
DNA junction
polynucleotide of a modified transgenic locus, in transgenic DNA of a DNA
junction
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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).
[0054] 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.
[0055] 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).
[0056] 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.
[0057] Various sequences set forth in the sequence listing are described in
the following table.
[0058] Table 1. Description of sequences.
SEQ ID NO Description
DAS81419 Complete Transgenic Locus comprising from 5' to 3': 5'
flanking plant genomic DNA; 5' junction polynucleotide; an AtUbil0
promoter; a CrylF coding sequence; an 0RF23 3' UTR; a CsYMV
promoter; a Cry lAc coding region; an 0RF23 3' UTR; a CsYMV promoter;
a PAT coding region; an ORF1 3' UTR; 3' junction polynucleotide, and 3'
flanking plant genomic DNA. The DA581419 5' flank region comprises
nucleotides 1-1400 of SEQ ID NO: 1. The DA581419 transgenic insert
extends from nucleotides 1401-13896 of SEQ ID NO: 1. The DAS81419 3'
1 flanking DNA comprises 13897-15294 of SEQ ID NO: 1.
INIR19-1 (with gRNA-1 Cut resulting in a deletion of nucleotides in a
2 DAS81419 3' junction polynucleotide sequence)
INIR19-2 (Insertion of 27 bp CgRRS of SEQ ID NO: 9 in 3' junction
polynucleotide with gRNA-2 Cut and SEQ ID NO: 23 donor DNA template
3 containing the SEQ ID NO: 9 CgRRS)
4 gRNA-1
gRNA-2
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6 gRNA-3
7 OgRRS
8 CgRRS+ flank DNA (G1 Insert)
9 CgRRS+ Flank DNA (G2 Insert)
CgRRS+ Flank DNA (G3 Insert)
11 DAS81419 donor template sequence containing SEQ ID NO: 8 CgRRS
12 DAS81419 3' target insertion site
13 DA581419 -gRNA coding sequence
14 DA581419 5' primer
DA581419 3' primer
16 DA581419 CgRRS and flank
INIR19-3 (Insertion of 27 bp CgRRS of SEQ ID NO: 8 with gRNA-1 Cut
and SEQ ID NO: 11 donor DNA template containing the SEQ ID NO: 8
17 CgRRS)
(Cas12a Nuclease) (>splU2UMQ6ICS12A ACISB CRISPR-associated
endonuclease Cas12a OS=Acidaminococcus sp. (strain BV3L6)
18 OX=1111120 GN=cas12a PE=1 SV=1)
19 DA581419 5' Junction Polynucleotide
DAS81419 5' plant genomic flanking
21 DA581419 3' Junction Polynucleotide
22 DAS81419 3' plant genomic flanking
23 INIR19-2 donor DNA template sequence containing SEQ ID NO: 9
CgRRS
[0059] 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. FEB S 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 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.
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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.
[0060] 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 1. In certain embodiments, INIR19
transgenic loci provided here
are characterized by polynucleotide sequences that can facilitate as necessary
the removal of the
INIR19 transgenic loci from the genome. Useful applications of such INIR19
transgenic loci and
related methods of making include targeted excision of a INIR19 transgenic
locus or portion
thereof in certain breeding lines to facilitate recovery of germplasm with
subsets of transgenic traits
tailored for specific geographic locations and/or grower preferences. Other
useful applications of
such INIR19 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 INIR19 transgenic loci or portions thereof which can be
selectively excised
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with one or more gRNA molecules and RdDe (RNA dependent DNA endonucleases)
which form
gRNA/target DNA complexes. Such selectively excisable INIR19 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
2A and B). In certain
embodiments, an originator guide RNA recognition site (OgRRS) comprises
endogenous DNA
found in untransformed plants and in endogenous non-transgenic DNA ofjunction
polynucleotides
of transgenic plants containing a modified or unmodified transgenic locus. In
certain embodiments,
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. Examples of OgRRS polynucleotide sequences in or near a 3'
junction
polynucleotide in an DAS81419 transgenic locus include SEQ ID NO: 7. 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 2A and as
elsewhere described
herein. A donor DNA template for introducing the SEQ ID NO: 7 OgRRS into the
3' junction
polynucleotide of an DA581419 locus includes the donor DNA template of SEQ ID
NO: 11 or 23.
Double stranded breaks in a 3' junction polynucleotide of SEQ ID NO: 1 can be
introduced with
gRNAs encoded by SEQ ID NO: 4, 5, or 6 and a Cas12a nuclease. Integration of
the SEQ ID NO:
11, 23, or other donor DNA template into the 3' junction polynucleotide of an
DA581419 locus at
the double stranded breaks introduced by the gRNAs encoded by SEQ ID NO: 4, 5,
and/or 6 and
a Cas12a nuclease can provide an INIR19 locus comprising the CgRRS sequence
set forth in SEQ
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ID NO: 8, 9, or 10. 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: 8. An INIR19 transgenic locus
containing this
CgRRS insertion of SEQ ID NO: 8 is set forth in SEQ ID NO: 17. A donor DNA
template of SEQ
ID NO: 23 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: 9. An INIR19
transgenic locus containing this CgRRS insertion of SEQ ID NO: 9 is set forth
in SEQ ID NO: 3.
[0061] Also provided are unique transgenic locus excision sites created
by excision of
INIR19 transgenic loci or selectively excisable INIR19 transgenic loci, DNA
molecules
comprising the INIR19 transgenic loci or unique fragments thereof (i.e.,
fragments of an INIR19
locus which are not found in an DAS81419 transgenic locus), INIR19 plants
comprising the same,
biological samples containing the DNA, nucleic acid markers adapted for
detecting the DNA
molecules, and related methods of identifying soybeanw plants comprising
unique INIR19
transgenic locus excision sites and unique fragments of a INIR19 transgenic
locus. An example of
such an excision site would include an excision site created by excising an
INIR19 transgenic locus
comprising a CgRRS of SEQ ID NO: 8, 9, or 10 located in a 3' junction
polynucleotide with a
guide RNA encoded by SEQ ID NO: 13 and a suitable Cas RdDe (e.g., a Cas12a
nuclease of SEQ
ID NO: 18). DNA molecules comprising unique fragments of an INIR19 transgenic
locus are
diagnostic for the presence of an INIR19 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 INIR19 transgenic
locus include
DNA molecules comprising SEQ ID NO: 8, 9, 10, 11, 16, 23, and allelic variants
therof.
[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 DAS81419
transgenic locus
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described in US Patent No. 8,680,363. Soybean plants comprising the DAS81419
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 DA581419 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
"b ch. cbd.int/database/organisms").
[0063] Sequences of the junction polynucleotides as well as the
transgenic insert(s) of the
DAS81419 transgenic locus which can be improved by the methods provided herein
are set forth
or otherwise provided in SEQ ID NO: 1, US 8,680,363, the sequence of the
DAS81419 locus in
the deposited seed of ATCC accession No. PTA-12006, and elsewhere in this
disclosure. In certain
embodiments provided herein, the DA581419 transgenic locus set forth in SEQ ID
NO: 1 or
present in the deposited seed of ATCC accession No. PTA-12006 is referred to
as an "original
DAS81419 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 DAS81419
transgenic plant
loci (e.g., progeny of deposited seed of accession No. PTA-12006 which contain
allelic variants of
SEQ ID NO:1 or progeny originating from transgenic plant cells comprising the
original
DA581419 transgenic loci set forth in US 8,680,363) 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, or 10,579 nucleotides of SEQ ID
NO: 1. Also
provided are plants, plant parts including seeds, genomic DNA, and/or DNA
obtained from INIR19
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 INIR19
transgenic locus or a
portion thereof Also provided herein are methods of detecting plants, genomic
DNA, and/or DNA
obtained from plants comprising a INIR19 transgenic locus which contains one
or more of a
CgRRS, deletions of selectable marker genes, deletions of non-essential DNA,
and/or a transgenic
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locus excision site. A first junction polynucleotide of a DAS81419 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 INIR19 transgenic locus. In certain embodiments, the OgRRS is found
in non-transgenic
DNA or transgenic DNA of the 5' junction polynucleotide of a transgenic locus
of any one of SEQ
ID NO: 1, 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 DA581419 transgenic locus of SEQ ID NO: 1,
allelic variants
thereof, or other variants thereof to obtain an INIR19 transgenic locus. In
other embodiments, the
OgRRS is found in non-transgenic DNA or transgenic DNA of the 3' junction
polynucleotide of
the DA581419 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 INIR19 transgenic
locus.
[0064] Also provided herein are allelic variants of any of the INIR19
transgenic loci or
DNA molecules provided herein. In certain embodiments, such allelic variants
of INIR19
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, 14,000, 15,000, or more nucleotides of SEQ ID NO: 2,
3, or 17. In certain
embodiments, such allelic variants of INIR19 DNA molecules include sequences
having at least
85%, 90%, 95%, 98%, or 99% sequence identity across the entire length of SEQ
ID NO: 2, 3, 8,
9, 10, 11, 16, 17, or 23.
[0065] 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
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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 INIR19 transgenic locus, in transgenic DNA of a DNA junction
polynucleotide of an INIR19
transgenic locus, or can span the junction of the transgenic and non-
transgenic DNA of a DNA
junction polynucleotide of an INIR19 transgenic locus. An OgRRS can likewise
be located in non-
transgenic plant genomic DNA of a DNA junction polynucleotide of an INIR19
transgenic locus,
in transgenic DNA of a DNA junction polynucleotide of an INIR19 transgenic
locus, or can span
the junction of the transgenic and non-transgenic DNA of a DNA junction
polynucleotide of an
INIR19 transgenic locus
[0066] Methods provided herein can be used in a variety of breeding
schemes to obtain
elite crop plants comprising subsets of desired modified transgenic loci
comprising an OgRRS and
a CgRRS operably linked to junction polynucleotide sequences and transgenic
loci excision sites
where undesired transgenic loci or portions thereof have been removed (e.g.,
by use of the OgRRS
and a CgRRS). Such methods are useful at least insofar as they allow for
production of distinct
useful donor plant lines each having unique sets of modified transgenic loci
and, in some instances,
targeted genetic changes that are tailored for distinct geographies and/or
product offerings. In an
illustrative and non-limiting example, a different product lines comprising
transgenic loci
conferring only two of three types of herbicide tolerance (e.g.., glyphosate,
glufosinate, and
dicamba) can be obtained from a single donor line comprising three distinct
transgenic loci
conferring resistance to all three herbicides. In certain aspects, plants
comprising the subsets of
undesired transgenic loci and transgenic loci excision sites can further
comprise targeted genetic
changes. Such elite crop plants can be inbred plant lines or can be hybrid
plant lines. In certain
embodiments, at least two transgenic loci (e.g., transgenic loci including an
INIR19 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
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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 1 (bottom
"Alternative" panel), where two or more of the transgenic loci ("Event" in
Figure 1) are provided
in Line A and then moved into elite crop plant germplasm by introgression. In
the non-limiting
Figure 1 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 1) comprising two or more of the modified
transgenic loci. This
elite germplasm containing the modified transgenic loci (e.g., "Universal
Donor" of Figure 1) can
then be subjected to genome editing molecules which can excise at least one of
the transgenic loci
("Event Removal" in Figure 1) and introduce other targeted genetic changes
("GE" in Figure 1) 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 1). 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 INIR19
transgenic loci
can be used as a pollen donor or recipient for hybrid seed production. Such
hybrid seed and the
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progeny grown therefrom can comprise a subset of desired transgenic loci and a
transgenic loci
excision site.
[0067] Hybrid plant lines comprising elite crop plant germplasm, at least
one transgenic
locus and at least one transgenic locus excision site, and in certain aspects,
additional targeted
genetic changes are also provided herein. Methods for production of such
hybrid seed can
comprise crossing elite crop plant lines where at least one of the pollen
donor or recipient comprises
at least the transgenic locus and a transgenic locus excision site and/or
additional targeted genetic
changes. In certain embodiments, the pollen donor and recipient will comprise
germplasm of
distinct heterotic groups and provide hybrid seed and plants exhibiting
heterosis. In certain
embodiments, the pollen donor and recipient can each comprise a distinct
transgenic locus which
confers either a distinct trait (e.g., herbicide tolerance or insect
resistance), a different type of trait
(e.g., tolerance to distinct herbicides or to distinct insects such as
coleopteran or lepidopteran
insects), or a different mode-of-action for the same trait (e.g., resistance
to coleopteran insects by
two distinct modes-of-action or resistance to lepidopteran insects by two
distinct modes-of-action).
In certain embodiments, the pollen recipient will be rendered male sterile or
conditionally male
sterile. Methods for inducing male sterility or conditional male sterility
include emasculation (e.g.,
detasseling), cytoplasmic male sterility, chemical hybridizing agents or
systems, a transgenes or
transgene systems, and/or mutation(s) in one or more endogenous plant genes.
Descriptions of
various male sterility systems that can be adapted for use with the elite crop
plants provided herein
are described in Wan et al. Molecular Plant; 12, 3, (2019):321-342 as well as
in US 8,618,358; US
20130031674; and US 2003188347.
[0068] 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 INIR19 transgenic locus
comprising an OgRRS in
non-transgenic DNA of a 1st 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 INIR19 transgenic locus excision site. For example, an
INIR19 transgenic
locus set forth in SEQ ID NO: 3 or 17 can be deleted with a Cas12a RdDe (e.g.
the Cas12a of SEQ
ID NO: 18) and a gRNA comprising an RNA encoded by SEQ ID NO: 13. In certain
embodiments,
the transgenic locus excision site can comprise a contiguous segment of DNA
comprising at least
base pairs of DNA that is telomere proximal to the deleted segment of the
transgenic locus and
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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 10 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
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
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
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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
INIR19 transgenic loci excision sites are provided herein.
[0069] In other embodiments, a segment comprising a INIR19 transgenic
locus (e.g., a
transgenic locus comprising an OgRRS in non-transgenic DNA of a 1" junction
sequence and a
CgRRS in a 2nd junction sequence) can be deleted with a gRNA and RdDe that
recognize the
OgRRS and the CgRRS and replaced with DNA comprising the endogenous non-
transgenic plant
genomic DNA present in the genome prior to transgene insertion. A non-limiting
example of such
replacements can be visualized in Figure 2C, 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.
[0070] In certain embodiments, edited transgenic plant genomes and
transgenic plant cells,
plant parts, or plants containing those edited genomes, comprising a
modification of an original
transgenic locus, where the modification comprises an OgRRS and a CgRRS which
are operably
linked to a 1" and a 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,
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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 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
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embodiments of an edited transgenic plant genome, the modification comprises a
modification of
a DAS81419 transgenic locus.
[0071] 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 INIR19 transgenic locus. In certain
embodiments, the
CgRRS/gRNA and OgRRS/gRNA hybridization complexes are recognized by the same
class of
RdDe (e.g., Class 2 type II or Class 2 type V) or by the same RdDe (e.g., both
the CgRRS/gRNA
and OgRRS/gRNA hybridization complexes recognized by the same Cas9 or Cas 12
RdDe). Such
CgRRS and OgRRS can be recognized by RdDe and suitable guide RNAs containing
crRNA
sufficiently complementary to the guide RNA hybridization site DNA sequences
adjacent to the
PAM site of the CgRRS and the OgRRS to provide for cleavage within or near the
two junction
polynucleotides. Suitable guide RNAs can be in the form of a single gRNA
comprising a crRNA
or in the form of a crRNA/tracrRNA complex. In the case of the OgRRS site, the
PAM and guide
RNA hybridization site are endogenous DNA polynucleotide molecules found in
the plant genome.
In certain embodiments where the CgRRS is introduced into the plant genome by
genome editing,
gRNA hybridization site polynucleotides introduced at the CgRRS are at least
17 or 18 nucleotides
in length and are complementary to the crRNA of a guide RNA. In certain
embodiments, the gRNA
hybridization site sequence of the OgRRS and/or the CgRRS is about 17 or 18 to
about 24
nucleotides in length. The gRNA hybridization site sequence of the OgRRS and
the gRNA
hybridization site of the CgRRS can be of different lengths or comprise
different sequences so long
as there is sufficient complementarity to permit hybridization by a single
gRNA and recognition
by a RdDe that recognizes and cleaves DNA at the gRNA/OgRRS and gRNA/CgRRS
complex. In
certain embodiments, the guide RNA hybridization site of the CgRRS comprise
about a 17 or 18
to about 24 nucleotide sequence which is identical to the guide RNA
hybridization site of the
OgRRS. In other embodiments, the guide RNA hybridization site of the CgRRS
comprise about a
17 or 18 to about 24 nucleotide sequence which has one, two, three, four, or
five nucleotide
insertions, deletions or substitutions when compared to the guide RNA
hybridization site of the
OgRRS. Certain CgRRS comprising a gRNA hybridization site containing has one,
two, three,
four, or five nucleotide insertions, deletions or substitutions when compared
to the guide RNA
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hybridization site of the OgRRS can undergo hybridization with a gRNA which is
complementary
to the OgRRS gRNA hybridization site and be cleaved by certain RdDe. Examples
of mismatches
between gRNAs and guide RNA hybridization sites which allow for RdDe
recognition and
cleavage include mismatches resulting from both nucleotide insertions and
deletions in the DNA
which is hybridized to the gRNA (e.g., Lin et al., doi: 10.1093/nar/gku402).
In certain
embodiments, an operably linked PAM site is co-introduced with the gRNA
hybridization site
polynucleotide at the CgRRS. In certain embodiments, the gRNA hybridization
site
polynucleotides are introduced at a position adjacent to a resident endogenous
PAM sequence in
the junction polynucleotide sequence to form a CgRRS where the gRNA
hybridization site
polynucleotides are operably linked to the endogenous PAM site. In certain
embodiments, non-
limiting features of the OgRRS, CgRRS, and/or the gRNA hybridization site
polynucleotides
thereof include: (i) absence of significant homology or sequence identity
(e.g., less than 50%
sequence identity across the entire length of the OgRRS, CgRRS, and/or the
gRNA hybridization
site sequence) to any other endogenous or transgenic sequences present in the
transgenic plant
genome or in other transgenic genomes of the 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
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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 create a double stranded break to insert or create a CgRRS, include
PAM sites recognized
by a Cas12a enzyme. Non-limiting examples where a CgRRS are created in a DNA
sequence are
illustrated in Example 2.
[0072] Transgenic loci comprising OgRRS and CgRRS in a first and a second
junction
polynucleotides can be excised from the genomes of transgenic plants by
contacting the transgenic
loci with RdDe or RNA directed nickases, and a suitable guide RNA directed to
the OgRRS and
CgRRS. A non-limiting example where a modified transgenic locus is excised
from a plant genome
by use of a gRNA and an RdDe that recognizes an OgRRS/gRNA and a CgRRS/gRNA
complex
and introduces dsDNA breaks in both junction polynucleotides and repaired by
NHEJ is depicted
in Figure 2B. In the depicted example set forth in Figure 2B, 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.
[0073] In certain embodiments, edited transgenic plant genomes provided
herein can lack
one or more selectable and/or scoreable markers found in an original event
(transgenic locus).
Original DAS81419 transgenic loci (events), including those set forth in SEQ
ID NO: 1), US
8,680,363, the sequence of the DAS81419 locus in the deposited seed of
accession No. PTA-12006
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 DAS81419 transgenic locus
is absent from an
INIR19 transgenic locus. The PAT selectable marker cassette can be excised
from an original
DA581419 transgenic locus by contacting the transgenic locus with one or more
gene editing
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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 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.
[0074] 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
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junction polynucleotides and providing the new expression cassettes on a donor
DNA template
(e.g., in Figure 2C, the donor DNA template can comprise an expression
cassette flanked by DNA
homologous to non-transgenic DNA located telomere proximal and centromere
proximal to the
excision site). Suitable expression cassettes for insertion include DNA
molecules comprising
promoters which are operably linked to DNA encoding proteins and/or RNA
molecules which
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.
[0075] In certain embodiments, INIR19 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 1, 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
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resistance (e.g., resistance to insect, nematode, bacterial, or fungal
damage), increased abiotic
stress resistance (e.g., resistance to drought, cold, heat, metal, or salt),
enhanced lodging resistance,
enhanced growth rate, enhanced biomass, enhanced tillering, enhanced
branching, delayed
flowering time, delayed senescence, increased flower number, improved
architecture for high
density planting, improved photosynthesis, increased root mass, increased cell
number, improved
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 example, 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,
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and the change in expression is upregulation or downregulation; (n) a hormone
responsive element
recognized by a nuclear receptor or a hormone-binding domain thereof; (o) a
transcription factor
binding sequence; and (p) a polycomb response element (see Xiao et al. (2017)
Nature Genetics,
49:1546-1552, doi: 10.1038/ng.3937). Non limiting examples of target 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).
[0076] In certain embodiments, it will be desirable to use genome editing
molecules to
make modified transgenic loci by introducing a CgRRS into the transgenic loci,
to excise modified
transgenic loci comprising an OgRRS and a CgRRS, and/or to make targeted
genetic changes in
elite crop plant or other germplasm. In certain embodiments, the genome edits
can be effected in
regenerable plant parts (e.g., plant embryos) of elite crop plants by
transient provision of gene
editing molecules or polynucleotides encoding the same and do not necessarily
require
incorporating a selectable marker gene into the plant genome (e.g., US
20160208271 and US
20180273960, both incorporated herein by reference in their entireties;
Svitashev et al. Nat
Commun. 2016; 7:13274).
[0077] 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,
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a nCas12a nickase, a Cas12d (CasY), a Cas12e (CasX), a Cas12b (C2c1), a Cas12c
(C2c3), a
Cas12i, a Cas12j, a Cas14, an engineered nuclease, a codon-optimized nuclease,
a zinc-finger
nuclease (ZEN) or nickase, a transcription activator-like effector nuclease
(TAL-effector nuclease
or TALEN) or nickase (TALE-nickase), an Argonaute, and a meganuclease or
engineered
meganuclease; (b) a polynucleotide encoding one or more nucleases capable of
effectuating site-
specific alteration (including introduction of a DSB or SSB) of a target
nucleotide sequence; (c) a
guide RNA (gRNA) for an RNA-guided nuclease, or a DNA encoding a gRNA for an
RNA-guided
nuclease; (d) donor DNA template polynucleotides; and (e) other DNA templates
(dsDNA,
ssDNA, or combinations thereof) suitable for insertion at a break in genomic
DNA (e.g., by non-
homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ).
[0078] 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) to form an RNA-
guided
endonuclease/guide RNA complex which can specifically bind sequences in the
gDNA target site
that are adjacent to a protospacer adjacent motif (PAM) sequence. The type of
RNA-guided
endonuclease typically informs the location of suitable PAM sites and design
of crRNAs or
sgRNAs. G-rich PAM sites, e.g., 5'-NGG are typically targeted for design of
crRNAs or sgRNAs
used with Cas9 proteins. Examples of PAM sequences include 5'-NGG
(Streptococcus pyogenes),
5' -NNAGAA (Streptococcus thermophilus CRISPR1), 5' -NGGNG (Streptococcus
thermophilus
CRISPR3), 5' -NNGRRT or 5'-NNGRR (Staphylococcus aureus Cas9, SaCas9), and 5' -
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NNNGATT (Neisseria meningitidis). T-rich PAM sites (e.g., 5'-TTN or 5'-TTTV,
where "V" is
A, C, or G) are typically targeted for design of crRNAs or sgRNAs used with
Cas12a proteins. In
some instances, Cas12a can also recognize a 5'-CTA PAM motif. Other examples
of potential
Cas12a PAM sequences include TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN, ATTN,
TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN, GCCN,
and CCGN (wherein N is defined as any nucleotide). Cpfl (i.e., Cas12a)
endonuclease and
corresponding guide RNAs and PAM sites are disclosed in US Patent Application
Publication
2016/0208243 Al, which is incorporated herein by reference for its disclosure
of DNA encoding
Cpfl endonucleases and guide RNAs and PAM sites. Introduction of one or more
of a wide variety
of CRISPR guide RNAs that interact with CRISPR endonucleases integrated into a
plant genome
or otherwise provided to a plant is useful for genetic editing for providing
desired phenotypes or
traits, for trait screening, or for gene editing mediated trait introgression
(e.g., for introducing a
trait into a new genotype without backcrossing to a recurrent parent or with
limited backcrossing
to a recurrent parent). Multiple endonucleases can be provided in expression
cassettes with the
appropriate promoters to allow multiple genome site editing.
[0079]
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
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the patent publications referenced in this paragraph are incorporated herein
by reference in their
entirety. In certain embodiments, an RNA-guided endonuclease that leaves a
blunt end following
cleavage of the target site is used. Blunt-end cutting RNA-guided
endonucleases include Cas9,
Cas12c, and Cas 12h (Yan et al., 2019). In certain embodiments, an RNA-guided
endonuclease
that leaves a staggered single stranded DNA overhanging end following cleavage
of the target site
following cleavage of the target site is used. Staggered-end cutting RNA-
guided endonucleases
include Cas12a, Cas12b, and Cas12e.
[0080] 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
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necessary for efficient DNA cleavage in vitro; see Zetsche et at. (2015) Cell,
163:759 ¨ 771. In
practice, guide RNA sequences are generally designed to have a length of 17 ¨
24 nucleotides
(frequently 19, 20, or 21 nucleotides) and exact complementarity (i.e.,
perfect base-pairing) to the
targeted gene or nucleic acid sequence; guide RNAs having less than 100%
complementarity to
the target sequence can be used (e.g., a gRNA with a length of 20 nucleotides
and 1 ¨4 mismatches
to the target sequence) but can increase the potential for off-target effects.
The design of effective
guide RNAs for use in plant genome editing is disclosed in US Patent
Application Publication
2015/0082478 Al, the entire specification of which is incorporated herein by
reference. More
recently, efficient gene editing has been achieved using a chimeric "single
guide RNA"
("sgRNA"), an engineered (synthetic) single RNA molecule that mimics a
naturally occurring
crRNA-tracrRNA complex and contains both a tracrRNA (for binding the nuclease)
and at least
one crRNA (to guide the nuclease to the sequence targeted for editing); see,
for example, Cong et
at. (2013) Science, 339:819 ¨ 823; Xing et at. (2014) BMC Plant Biol., 14:327
¨ 340. Chemically
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.
[0081] 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
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Aug; 3(8):eaa04774. In other embodiments, adenine base editors (ABEs)
comprising an adenine
deaminase fused to catalytically inactive Cas9 (dCas9) or a Cas9 D 1 OA
nickase can be used to
convert A/T base pairs to G/C base pairs in genomic DNA (Gaudelli et al.,
(2017) Nature
551(7681):464-471.
[0082] 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
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incorporated herein by reference in their entirety. Exemplary selection
methods (e.g., phage display
and yeast two-hybrid systems) can be adapted for use in the methods described
herein. In addition,
enhancement of binding specificity for zinc finger binding domains has been
described in US
Patent 6,794,136, incorporated herein by reference in its entirety. In
addition, individual zinc finger
domains may be linked together using any suitable linker sequences. Examples
of linker sequences
are publicly known, e.g., see US Patents 6,479,626; 6,903,185; and 7,153,949,
incorporated herein
by reference in their entirety. The nucleic acid cleavage domain is non-
specific and is typically a
restriction endonuclease, such as Fokl. This endonuclease must dimerize to
cleave DNA. Thus,
cleavage by Fokl as part of a ZFN requires two adjacent and independent
binding events, which
must occur in both the correct orientation and with appropriate spacing to
permit dimer formation.
The requirement for two DNA binding events enables more specific targeting of
long and
potentially unique recognition sites. Fokl variants with enhanced activities
have been described
and can be adapted for use in the methods described herein; see, e.g., Guo et
at. (2010)1 Mol.
Biol., 400:96 - 107.
[0083] Transcription activator like effectors (TALEs) are proteins
secreted by certain
Xanthomonas species to modulate gene expression in host plants and to
facilitate the colonization
by and survival of the bacterium. TALEs act as transcription factors and
modulate expression of
resistance genes in the plants. Recent studies of TALEs have revealed the code
linking the
repetitive region of TALEs with their target DNA-binding sites. TALEs comprise
a highly
conserved and repetitive region consisting of tandem repeats of mostly 33 or
34 amino acid
segments. The repeat monomers differ from each other mainly at amino acid
positions 12 and 13.
A strong correlation between unique pairs of amino acids at positions 12 and
13 and the
corresponding nucleotide in the TALE-binding site has been found. The simple
relationship
between amino acid sequence and DNA recognition of the TALE binding domain
allows for the
design of DNA binding domains of any desired specificity. TALEs can be linked
to a non-specific
DNA cleavage domain to prepare genome editing proteins, referred to as TAL-
effector nucleases
or TALENs. As in the case of ZFNs, a restriction endonuclease, such as Fokl,
can be conveniently
used. Methods for use of TALENs in plants have been described and can be
adapted for use in the
methods described herein, see Mahfouz et al. (2011) Proc. Natl. Acad. Sci.
USA, 108:2623 ¨2628;
Mahfouz (2011) GM Crops, 2:99 ¨ 103; and Mohanta et al. (2017) Genes vol.
8,12: 399). TALE
nickases have also been described and can be adapted for use in methods
described herein (Wu et
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al.; Biochem Biophys Res Commun. (2014);446(1):261-6; Luo et al; Scientific
Reports 6,
Article number: 20657 (2016)).
[0084] 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 nuclease effects an
off-set DSB with 5-nucleotide overhangs in the genomic sequence, the donor DNA
template
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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 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
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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 or 23 and equivalents thereof with longer or
shorter homology arms.
In certain embodiments, a donor DNA template can comprise an adapter molecule
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.
[0085] Donor DNA template molecules used in the methods provided herein
include DNA
molecules comprising, from 5' to 3', a first homology arm, a replacement DNA,
and a second
homology arm, wherein the homology arms containing sequences that are
partially or completely
homologous to genomic DNA (gDNA) sequences flanking a target site-specific
endonuclease
cleavage site in the gDNA. In certain embodiments, the replacement DNA can
comprise an
insertion, deletion, or substitution of 1 or more DNA base pairs relative to
the target gDNA. In an
embodiment, the donor DNA template molecule is double-stranded and perfectly
base-paired
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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 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
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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.
[0086] Various treatments are useful in delivery of gene editing
molecules and/or other
molecules to a DAS81419 or INIR19 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 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.,
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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.
[0087] 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 DAS81419 or INIR19
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, 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
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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, 7SL (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 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.
[0088] 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
"polyadenylation signal."
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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.
[0089]
In certain embodiments, the DA581419 or INIR19 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 ages/d/d3/Hapl oi d Arab i dop si
s_protocol [dot] p df; (Ravi et at.
(2014) Nature Communications, 5:5334, doi: 10.1038/nc0mm56334). Haploids can
also be
obtained in a wide variety of monocot plants (e.g., 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,
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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.
[0090] In certain embodiments, the DAS81419 or INIR19 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.
[0091] In certain embodiments, the DAS81419 or INIR19 plant cells in used
in the methods
provided herein can include dividing cells. Dividing cells can include those
cells found in various
plant tissues including leaves, meristems, and embryos. These tissues include
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.
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[0092] In some embodiments, methods provided herein can include the
additional step of
growing or regenerating an INIR19 plant from a INIR19 plant cell that had been
subjected to the
gene editing or from a regenerable plant structure obtained from that INIR19
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 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
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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
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 INIR19
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
[0093] Various embodiments of the plants, genomes, methods, biological
samples, and
other compositions described herein are set forth in the following sets of
numbered embodiments.
[0094] la. A transgenic soybean plant cell comprising an INIR19
transgenic locus
comprising an originator guide RNA recognition site (OgRRS) in a first DNA
junction
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polynucleotide of a DAS81419 transgenic locus and a cognate guide RNA
recognition site
(CgRRS) in a second DNA junction polynucleotide of the DAS81419 transgenic
locus.
[0095] lb. A transgenic soybean plant cell comprising an INIR19
transgenic locus
comprising an insertion and/or substitution of DNA in a DNA junction
polynucleotide of a
DAS81419 transgenic locus with DNA comprising a cognate guide RNA recognition
site (CgRRS)
or comprising a deletion in a DNA junction polynucleotide of a DAS81419
transgenic locus.
[0096] 2. The transgenic soybean plant cell of embodiment la or lb,
wherein said CgRRS
comprises the DNA molecule set forth in SEQ ID NO: 8, 9, or 10; and/or wherein
said DAS81419
transgenic locus is set forth in SEQ ID NO:1, is present in seed deposited at
the ATCC under
accession No. PTA-12006, is present in progeny thereof, is present in allelic
variants thereof, or is
present in other variants thereof.
[0097] 3. The transgenic soybean plant cell of embodiments la, lb, or 2,
wherein said
INIR19 transgenic locus comprises the DNA molecule set forth in SEQ ID NO: 2,
3, 17, or an
allelic variant thereof.
[0098] 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.
[0099] 5. A transgenic soybean plant comprising the soybean plant cell of
any one of
embodiments la, lb, 2, or 3.
[00100] 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 INIR19
transgenic locus from the selfed soybean plant.
[00101] 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 INIR19 transgenic locus
from the cross.
[00102] 8. A DNA molecule comprising SEQ ID NO: 2, 3, 8, 9, 10, 11, 17,
23, or an allelic
variant thereof.
[00103] 9. A processed transgenic soybean plant product comprising the DNA
molecule of
embodiment 8.
[00104] 10. A biological sample containing the DNA molecule of embodiment
8.
[00105] 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.
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[00106] 12. A method of detecting a soybean plant cell comprising the
INIR19 transgenic
locus of any one of embodiments la, lb, 2, or 3, comprising the step of
detecting DNA molecule
comprising SEQ ID NO: 2, 3, 8, 9, 10, 11, 17, 23, or an allelic variant
thereof.
[00107] 13. A method of excising the INIR19 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 INIR19 transgenic locus comprising the OgRRS and the CgRRS
with: (i) an RNA dependent DNA endonucl ease (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 INIR19 transgenic locus flanked by the OgRRS and the CgRRS has
been excised.
[00108] 14. The method of embodiment 13, wherein said INIR19 transgenic
locus
comprises the CgRRS in SEQ ID NO: 8, 9, or 10 and the guide RNA comprises an
RNA sequence
encoded by SEQ ID NO: 13.
[00109] 15. The method of embodiment 14, wherein said INIR19 transgenic
locus
comprises the DNA molecule set forth in SEQ ID NO: 17, 3, or an allelic
variant thereof.
Examples
1001101 Example 1. Introduction of a CgRRS in a 3' junction polynucleotide
of a
DAS81419 Transgenic Locus
[00111] Transgenic plant genomes containing one or more of the following
transgenic loci
(events) are contacted with:
(i) an ABE or CBE and guide RNAs which recognize the indicated target DNA
sites (protospacer
(guide RNA coding) plus PAM site) in the 5' or3' junction polynucleotides of
the event to
introduce a CgRRS in the junction polynucleotide;
(ii) an RdDe and guide RNAs which recognize the indicated target DNA site
(guide RNA coding
plus PAM site) in the 5' or 3' junction polynucleotides of the event as well
as a donor DNA
template spanning the double stranded DNA break site in the junction
polynucleotide to introduce
a CgRRS in a junction polynucleotide.
Plant cells, callus, parts, or whole plants comprising the introduced CgRRS in
the transgenic plant
genome are selected.
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[00112] Table 2. Examples of OgRRS and CgRRS in DAS81419
SOYBEAN
EVENT OgRRS CgRRS
NAME
DAS81419 (SEQ ID NO: 7; located (SEQ ID NO: 8; inserted into 3' junction
in 5' junction polynucleotide)
polynucleotide of SEQ ID
NO: 1)
(SEQ ID NO: 9; inserted into 3' junction
polynucleotide)
(SEQ ID NO: 10; inserted into 3'
junction polynucleotide)
[00113] Example 2. Insertion of a CgRRS element in the 3'-junction of the
DA581419
event.
[00114] 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 a OsUBIl, ZmUBIl, OsACT
promoter and a
OsUbil, ZmUBIl, OsACT polyadenylation site respectively, to provide the
exonuclease, SSAP,
and SSB plant expression cassettes.
[00115] A DNA donor template sequence (SEQ ID NO: 11) that targets the 3'-
T-DNA
junction polynucleotide of the DA581419 event (SEQ ID NO:1) for HDR-mediated
insertion of a
base pair OgRRS sequence (SEQ ID NO: 7) that is identical to a Cas12a
recognition site at the 5'-
junction polynucleotide of the DAS81419 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. The homology arms match (i.e., are homologous to) gDNA
(genomic
DNA) regions flanking the target gDNA insertion site (SEQ ID NO: 12). The
replacement template
region comprising the donor DNA is flanked at each end by DNA sequences
identical to the
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DAS81419 3'-T-DNA junction polynucleotide sequence recognized by a Cas12a RNA-
guided
nuclease and a gRNA (e.g., encoded by SEQ ID NO: 4, 5, or 6).
[00116] In the Agrobacterium approach, a binary vector that contains a
strong constitutive
expression cassette like the AtUbil0 promoter: :AtUbil0 terminator driving
Cas12a, a PolII or PolII
gene cassette driving the Cas12a gRNA(s) and a CaMV 355:NPTII:NOS (i.e., which
confers
tolerance to G418, kanamycin, and neomycin) 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.
In the example below, selections for the NPTII marker are provided. It is
understood that other
selection agents would be used for other selectable markers.
[00117] 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: 4, 5, or 6)
complementary to
sequences adjacent to the insertion site is constructed. An Agrobacterium
superbinary plasmid
transformation vector containing a cassette that provides for the expression
of the neomycin
phosphotransferase(nptII) protein 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.
[00118] A soybean transformation plasmid is constructed with the nptII or
hpt cassette, the
RNA-guided sequence-specific endonuclease cassette, the guide RNA cassette,
and the DAS81419
3'-T DNA junction sequence DNA donor sequence into the Agrobacterium
superbinary plasmid
transformation vector (the control vector).
[00119] A soybean transformation plasmid is constructed with the nptII or
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 DAS81419 3'-T DNA
junction sequence
donor DNA template sequence (e.g., SEQ ID NO: 11) into the Agrobacterium
superbinary plasmid
transformation vector (the lambda red vector).
[00120] The T-DNA-based expression cassettes are delivered from
superbinary vectors in
Agrobacterium strain LBA4404. Soy transformations are performed based on
published methods
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(e.g., Zhang et al., 1999, Plant Cell, Tissue and Organ Culture 56(1), 37-46)
or their equivalents.
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.
[00121] 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' (F17T8/750 cool white bulbs,
Litetronics(9). The co-
cultivation plates are wrapped with Parafilm .
[00122] 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
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
tmols-1 m2.
[00123] 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
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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 mg1-1NAA without
further selection
in Magenta boxes .
[00124]
[00125] When a sufficient amount of viable tissue is obtained, it can be
screened for
insertion at the DAS81419 junction sequence, using a PCR-based approach. The
PCR primer on
the 5'-end is SEQ ID NO: 14. The PCR primer on the 3'-end is SEQ ID NO: 15.
The above primers
that flank donor DNA homology arms are used to amplify the DA581419 3'-
junction
polynucleotide sequence. The correct donor sequence insertion will produce PCR
product (SEQ
ID NO: 16). A unique DNA fragment comprising the CgRRS in the DA581419 3'
junction
polynucleotide is set forth in SEQ ID NO: 8 and 16. 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 DA581419 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 INIR19 transgenic locus (SEQ ID NO: 17) comprising the CgRRS and
OgRRS (e.g.,
which each comprise SEQ ID NO: 7) can be excised using Cas12a and a suitable
gRNA which
hybridizes to DNA comprising SEQ ID NO: 13 at both the OgRRS and the CgRRS.
[00126] The breadth and scope of the present disclosure should not be
limited by any of the
above-described embodiments.
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