CN116367714A

CN116367714A - Genome editing of transgenic crop plants with modified transgene loci

Info

Publication number: CN116367714A
Application number: CN202180057942.1A
Authority: CN
Inventors: 迈克尔·安德烈亚斯·考克; 迈克尔·李·努奇奥; 弗雷德里克·范·埃克斯; 亚历山德拉·埃拉塔; 丹尼尔·罗德里格斯·莱尔; 约书亚·L·普莱斯
Original assignee: Inari Agricultural Technology Co ltd
Current assignee: Inari Agricultural Technology Co ltd
Priority date: 2020-07-31
Filing date: 2021-07-26
Publication date: 2023-06-30
Also published as: EP4172343A1; CA3188283A1; BR112023001759A2

Abstract

Methods for selectively excision of selectable marker genes and/or non-essential DNA from transgenic loci in transgenic plants and for producing targeted genetic alterations using genome editing techniques are provided. Transgenic plants comprising a transgenic locus lacking the selectable marker and/or non-essential DNA are also provided, and the use of such methods and plants for promoting plant breeding is disclosed.

Description

Genome editing of transgenic crop plants with modified transgene loci

Sequence listing

A sequence listing contained in a file named "10075wo1_st25.txt", measured 486,495 bytes in the Windows operating system, created at 14, 7, 2021 and submitted electronically via EFS-Web at 26, 7, 2021, which is incorporated herein by reference in its entirety.

Background

Transgenes placed at different locations in the plant genome by non-site specific integration may exhibit different levels of expression (Weising et al, 1988, ann.Rev.Genet. [ annual genetics ] 22:421-477). Such transgene insertion sites may also contain various undesirable rearrangements of foreign DNA elements, including deletions and/or duplications. In addition, many transgene insertion sites may also contain selectable or scorable marker genes, which in some cases are not required once a transgenic plant event is selected that contains the linked transgene conferring the desired trait.

Commercial transgenic plants typically contain one or more independent transgene insertions at specific locations in the host plant genome, which locations have been selected for characteristics including: expression of one or more transgenes of interest and one or more traits conferred by the transgenes, deletions or minimal rearrangements, and normal mendelian transmission of the one or more traits to the offspring. Examples of selected transgenic corn, soybean, cotton, and canola plant events that confer traits such as herbicide tolerance and/or pest tolerance are disclosed in U.S. patent No. 7323556;8575434;6040497;10316330;8618358;8212113;9428765;8455720;7897748;8273959;8093453;8901378;8466346; RE44962;9540655;9738904;8680363;8049071;9447428;9944945;8592650;10184134;7179965;7371940;9133473;8735661;7381861;8048632; and 9738903.

Methods for removing selectable marker genes and/or duplication transgenes at transgene insertion sites in plant genomes, which involve the use of site-specific recombinase systems (e.g., cre-lox) and insertion of new genes into transgene insertion sites, have been disclosed (Srivastava and Ow; methods Mol Biol [ Methods of molecular biology ],2015,1287:95-103; dale and Ow,1991,Proc.Natl Acad.Sci.USA [ Proc. Natl. Acad. Sci. USA ]88,10558-10562; srivastava and Thomson, plant Biotechnol J [ J. Biotechnology of plants ],2016;14 (2): 471-82). Such methods typically require the incorporation of recombination site sequences recognized by a recombinase at specific locations within the transgene.

Disclosure of Invention

Provided herein are methods of producing a elite crop plant comprising at least one modification of an approved transgene locus and at least one targeted genetic alteration conferring a desired trait, comprising the steps of: (a) transforming a plant cell or plant tissue of a elite crop plant with a marker-based transformation system comprising at least one selectable marker and one or more transgenes encoding at least one genome editing molecule, wherein the elite crop plant comprises a modification of the approved transgene locus, the modification comprising a deletion of a segment consisting essentially of, or consisting of, a selectable marker gene, and wherein the genome editing molecule is designed to induce the at least one targeted genetic alteration, (b) selecting a plant comprising stable integration of the transformation system in genomic DNA of the plant by utilizing the selectable marker, and (c) selecting a plant comprising the modification of the approved transgene locus and at least one targeted genetic alteration conferring a desired trait.

Provided herein are methods of producing a elite crop plant comprising at least one modification of a transgene locus conferring a targeted genetic alteration and approval of a desired trait, comprising the steps of: (i) Inducing at least one targeted genetic change in the maize plant genome with one or more genome editing molecules in a modified elite crop plant comprising the approved transgenic locus; and (ii) selecting a modified elite crop plant comprising the approved transgene locus, wherein the targeted genetic alteration.

Provided herein are elite crop plants comprising a modification of an original approved transgene locus, wherein the modification comprises a deletion of a segment comprising, consisting essentially of, or consisting of a selectable marker gene of the original approved transgene locus, and wherein the modification of the approved transgene locus does not comprise a site specific recombination system DNA recognition site.

Methods for obtaining elite crop plants are provided, comprising the steps of: (a) Obtaining a modified crop plant comprising the original approved transgene locus, the modification comprising a deletion of a selectable marker gene, consisting essentially of, or a segment consisting of the original approved transgene locus, wherein the plant does not comprise the germplasm of a elite crop plant; and (b) introgressing the modified transgenic locus into the germplasm of the elite crop plant.

Methods of obtaining a large population of inbred seeds for commercial seed production are provided, these comprising selfing the elite crop plants provided herein and harvesting seeds from the selfed elite crop plants.

Methods of obtaining hybrid seed are provided, comprising crossing a first plant comprising an edited transgenic plant genome comprising a modified transgenic locus described herein and optionally targeting a genetic alteration with a second plant and harvesting seed from the crossing.

DNA comprising selectable marker gene excision sites is provided in which a selectable marker gene, consisting essentially of, or a segment consisting of the original approved transgene locus is deleted.

A nucleic acid marker suitable for detecting genomic DNA or a fragment comprising a selectable marker gene excision site is provided, wherein a selectable marker gene comprising, consisting essentially of, or consisting of a primary approved transgene locus is deleted, and wherein the nucleic acid marker does not detect the primary approved transgene locus wherein the selectable marker gene is not deleted.

A biological sample is provided comprising plant genomic DNA or a fragment thereof comprising a selectable marker gene excision site wherein a selectable marker gene comprising, consisting essentially of, or a segment consisting of the original approved transgene locus is deleted.

Methods of identifying elite crop plants, DNA or biological samples described herein are provided, comprising detecting a polynucleotide comprising a selectable marker gene excision site with a nucleic acid detection assay, wherein a selectable marker gene comprising, consisting essentially of, or consisting of a segment of the original approved transgene locus is deleted.

Methods of enhancing the function of an approved transgenic locus are provided, the methods comprising deleting a segment of the original approved transgenic locus with one or more gene editing molecules, the segment comprising, consisting essentially of, or consisting of: replication of the transgene; replication of the transgenic element; and/or a fragment of the transgene; optionally, wherein the replication and/or fragment of the transgenic element is a promoter or a replication and/or fragment of a polyadenylation signal.

Drawings

FIG. 1 shows a schematic representation of the transgene expression cassette and selectable marker in the DAS-59122-7 transgene locus shown in SEQ ID NO. 1.

FIG. 2 shows a schematic representation of the transgene expression cassette and selectable marker in the DP-4114 transgene locus shown in SEQ ID NO. 2.

FIG. 3 shows a schematic representation of the transgene expression cassette and selectable marker in the MON87411 transgene locus shown in SEQ ID NO. 3.

FIG. 4 shows a schematic representation of the transgene expression cassette in the MON89034 transgene locus.

FIG. 5 shows a schematic representation of the transgene expression cassette and selectable marker in the MIR162 transgene locus.

FIG. 6 shows a schematic representation of the transgene expression cassette and selectable marker in the MIR604 transgene locus shown in SEQ ID NO. 6.

FIG. 7 shows a schematic representation of the transgene expression cassette and selectable marker in the NK603 transgene locus shown in SEQ ID NO. 7.

FIG. 8 shows a schematic representation of the transgene expression cassette and selectable marker in the SYN-E3272-5 transgene locus shown in SEQ ID NO. 8.

FIG. 9 shows a schematic representation of the transgene expression cassette and selectable marker in the TC1507 transgene locus shown in SEQ ID NO. 8.

FIG. 10 shows a schematic representation of the transgene expression cassette and selectable marker in the 5307 transgene locus shown in SEQ ID NO. 10.

FIG. 11 shows a schematic diagram comparing current transgenic event (i.e., transgenic locus) introgression breeding strategies with alternative breeding strategies for transgenic event introgression, wherein the transgenic event (i.e., transgenic locus) can be removed after introgression to provide a different combination of transgenic traits.

FIG. 12 shows a schematic representation of the transgene expression cassette and selectable marker in the DAS68416-4 transgene locus shown in SEQ ID NO. 12.

FIG. 13 shows a schematic representation of the transgene expression cassette and selectable marker in the MON87701 transgene locus shown in SEQ ID NO. 14.

FIG. 14 shows a schematic representation of the transgene expression cassette and selectable marker in the MON89788 transgene locus shown in SEQ ID NO. 16.

FIG. 15 shows a schematic representation of the transgene expression cassette and selectable marker in the COT102 transgene locus shown in SEQ ID NO. 19.

FIG. 16 shows a schematic representation of the transgene expression cassette and selectable marker in the MON88302 transgene locus shown in SEQ ID NO. 21.

Detailed Description

Unless otherwise indicated, nucleic acid sequences in the context of the present specification are given in the 5 'to 3' direction when read from left to right. The nucleic acid sequence may be provided in the form of DNA or RNA, as specifically described; as known to those of ordinary skill in the art, the disclosure of one necessarily defines the other, and necessarily defines the exact complement.

Where a term is provided in the singular, the inventors also contemplate embodiments described by the plural of that term.

The term "about" as used herein refers to a value or range of values, which may be understood as equivalents of the stated value, and may be greater than or less than 10% of the stated value or range of values. Each value or range of values beginning with the term "about" is also intended to cover embodiments of the stated absolute value or range of values.

The phrase "allelic variant" as used herein refers to a polynucleotide or polypeptide sequence variant that occurs in a different strain, variant, or isolate of a given organism.

The term "and/or" as used herein is to be taken as a specific disclosure of each of two specified features or components with or without another specified feature or component. Thus, the term "and/or" as used in phrases such as "a and/or B" herein is intended to include "a and B", "a or B", "a" (alone) and "B" (alone). Likewise, the term "and/or" as used in phrases 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).

As used herein, the phrase "approved transgenic locus" refers to a transgenic plant event that has been authorized, approved, and/or deregulated by a government machine for any of field trials, cultivation, human consumption, animal consumption, and/or import. Illustrative and non-limiting examples of government agencies that provide such approval include the Argentina department of agriculture, the New Zealand food Standard agency of Australia, the Brazilian national biosafety technical Committee (CTNBio), the Canada food inspection agency, the China department of agriculture biosafety network, the European food safety agency, the United states department of agriculture, the United states environmental protection agency, and the United states food and drug administration.

As used herein, the term "backcross" refers to crossing one or more F1 plants with one of the original parents. Backcrossing is used to maintain or establish the identity of one parent (species) and incorporate a specific trait of a second parent (species). As used herein, the term "backcross generation" refers to the offspring of a backcross.

As used herein, the phrase "biological sample" refers to whole or non-whole (e.g., ground seed or plant tissue, minced plant tissue, lyophilized tissue) plant tissue. It may also be an extract comprising whole or incomplete seeds or plant tissue. Biological samples may include flours, meal, syrups, oils, starches, and grains, all or part of which are manufactured to contain crop plant by-products. In certain embodiments, the biological sample is "non-renewable" (i.e., cannot be regenerated into plants or plant parts). In certain embodiments, a biological sample refers to a homogenate, extract, or any portion thereof containing genomic DNA of an organism from which the biological sample was obtained, wherein the biological sample does not comprise living cells.

For example, the terms "corresponding," "corresponding," and the like, when used in the context of nucleotide positions, mutations, and/or substitutions of any given polynucleotide (e.g., allelic variants of SEQ ID NOS: 1-34) relative to a reference polynucleotide sequence (e.g., SEQ ID NOS: 1-34), all refer to positions of a polynucleotide residue in a given sequence that have identity to a residue in a reference nucleotide sequence when the given polynucleotide is aligned to the reference polynucleotide sequence using a pairwise alignment algorithm (e.g., CLUSTAL O1.2.4 with default parameters).

As used herein, the terms "Cpf1" and "Cas12a" are used interchangeably to refer to the same RNA-dependent DNA endonuclease (RdDe). Cas12a proteins include the proteins provided herein as SEQ ID NO. 149.

As used herein, the term "crossing" refers to the fertilization of a female plant (or gamete) with a male plant (or gamete). The term "gamete" refers to a haploid germ cell (egg or pollen) in a plant where gametophytes are produced by meiosis and participate in sexual reproduction (in the process, two heterologous gametes fuse to form a diploid gamete). The term generally includes references to pollen (including sperm cells) and ovules (including ova). When referring to crosses in the context of effecting introgression of a genomic region or segment, the skilled artisan will understand that, in order to effect introgression of only a portion of the chromosome of one plant into the chromosome of another plant, random portions of the two parental line genomes recombine during the crossing due to crossover events occurring during gamete production of the parental line. Thus, the genomes of both parents must be combined by crossing in one cell, where the cell produces gametes and fusion during fertilization will result in an introgression event.

As used herein, the phrases "DNA-linked polynucleotide" and "linked polynucleotide" refer to polynucleotides from about 18 to about 500 base pairs in length that are made up of endogenous chromosomal DNA of the plant genome and heterologous transgenic DNA inserted into the plant genome. Thus, the joining polynucleotide may comprise about 8, 10, 20, 50, 100, 200, or 250 base pairs of plant genomic endogenous chromosomal DNA and about 8, 10, 20, 50, 100, 200, or 250 base pairs of heterologous transgenic DNA spanning one end of the transgene insertion site in the plant chromosomal DNA. The transgene insertion site in the chromosome typically comprises both a 5 'linked polynucleotide and a 3' linked polynucleotide. In the examples shown in SEQ ID NOS.1-34 herein, the 5 'joining polynucleotide is located at the 5' end of the sequence and the 3 'joining polynucleotide is located at the 3' end of the sequence.

The term "donor" as used herein in the context of a plant refers to a plant or plant line of trait, transgenic event or genomic segment origin, wherein the donor may have the trait, introgression or genomic segment in a heterozygous or homozygous state.

As used herein, the terms "excision" and "deletion" are used interchangeably in the context of a DNA molecule, and refer to the removal of a given DNA segment or element (e.g., a transgenic element) of the DNA molecule.

As used herein, the phrase "elite crop plant" refers to a plant that has been bred to provide improvement in one or more traits. Elite crop plant lines include substantially homozygous plants, e.g., inbred or doubled haploids. Good crop plants may include inbred lines used as such or as pollen donors or pollen acceptors in hybrid seed production (e.g. for the production of F1 plants). Elite crop plants may include inbreds that produce non-hybrid cultivars or varieties or that produce (e.g., increase in batches) pollen donor or acceptor lines for hybrid seed production. The elite crop plant may comprise hybrid F1 progeny crossed between two different elite inbred lines or doubled haploid plant lines.

As used herein, "event," "transgenic locus," and related phrases refer to the insertion of one or more transgenes at unique sites in the plant genome, as well as to DNA fragments, plant cells, plants, and plant parts (e.g., seeds, leaves, tubers, stems, roots, or pods) comprising genomic DNA containing the transgene insertion. Such events typically comprise both 5 'and 3' dna linked polynucleotides and confer one or more useful traits, including herbicide tolerance, insect resistance, male sterility, and the like.

As used herein, the phrases "endogenous sequence," "endogenous gene," "endogenous DNA," and the like refer to the native form of a polynucleotide, gene, or polypeptide in its native location in an organism or organism's genome.

As used herein, the term "exogenous DNA sequence" is any nucleic acid sequence that has been removed from its natural location and inserted into a new location, thereby altering the sequences flanking the moved nucleic acid sequence. For example, the exogenous DNA sequence may comprise a sequence from another species.

As used herein, the term "F1" refers to any offspring that are crossed between two genetically distinct individuals.

As used herein, the term "gene" refers to a genetic unit consisting of a DNA sequence that occupies a particular location on a chromosome and contains genetic instructions for a particular feature or trait in an organism. Thus, the term "gene" includes a nucleic acid (e.g., DNA or RNA) sequence that comprises the coding sequences necessary to produce RNA or a polypeptide or precursor thereof. The functional polypeptide may be encoded by the full-length coding sequence or by any portion of the coding sequence, so 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.

The term "identify" as used herein with respect to a plant refers to a process of establishing the identity or distinguishing traits of a plant, including exhibiting a trait, containing one or more transgenes, and/or containing one or more molecular markers.

The term "isolated" as used herein means having been removed from its natural environment.

As used herein, the term "comprising" should be interpreted as having at least the characteristics to which they refer, without excluding any other unspecified characteristics.

As used herein, the phrase "introduced transgene" is a transgene that is not present in the original transgene locus in the genome of the original transgenic event or in the genome of a progeny line obtained from the original transgenic event. Examples of transgenes introduced include exogenous transgenes inserted into a resident original transgene locus.

As used herein, the terms "introgression," "introgression," and "introgression" refer to natural and artificial processes, and plants produced thereby, in which a trait, gene, or DNA sequence is moved from one species, variety, or cultivar to another species, variety, or cultivar by crossing the species, variety, or cultivar with the other species, variety, or cultivar. This process may optionally be accomplished by backcrossing to the recurrent parent. Examples of introgression include the entry or introduction of a gene, transgene, regulatory element, marker, trait locus or chromosome segment from the genome of one plant into the genome of another plant.

As used herein, the phrase "marker assisted selection" refers to the following diagnostic process: plants are then selected from a group of plants, optionally using the presence of the molecular marker as a diagnostic feature or selection criterion. This process typically involves detecting the presence of a particular nucleic acid sequence or polymorphism in the plant genome.

As used herein, the phrase "molecular marker" refers to an indicator in a method for visualizing differences in nucleic acid sequence characteristics. Examples of such indicators include Restriction Fragment Length Polymorphism (RFLP) markers, amplified Fragment Length Polymorphism (AFLP) markers, single Nucleotide Polymorphisms (SNPs), microsatellite markers (e.g., SSRs), sequence Characterization Amplified Region (SCAR) markers, next Generation Sequencing (NGS) molecular markers, cut Amplified Polymorphic Sequence (CAPS) markers, or isozymic markers, or combinations of markers defining specific genetic and chromosomal locations as described herein.

As used herein, the term "natural" or "natural" defines conditions found in nature. A "native DNA sequence" is a DNA sequence that exists in nature and is produced by natural means or conventional breeding techniques, rather than by genetic engineering (e.g., using molecular biology/transformation techniques).

As used herein, the term "progeny" refers to any progeny produced by crosses, selfs, or other propagation techniques.

The phrase "operatively linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. When the phrase "operably linked" is used in the context of a PAM site and a DNA segment, it refers to a characteristic PAM site that allows cleavage of at least one DNA strand in the DNA segment with an RNA-dependent DNA endonuclease, an RNA-dependent DNA binding protein, or an RNA-dependent DNA nickase that recognizes the PAM site when there is a guide RNA complementary to the sequence adjacent to the PAM site.

As used herein, the term "plant" includes whole plants as well as any progeny, cells, tissues or parts of plants. The term "plant part" includes any one or more parts of a plant, including for example, but not limited to: seeds (including mature seeds and immature seeds); plant cuttings; a plant cell; plant cell cultures; or plant organs (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, and explants). The plant tissue or plant organ may be a seed, a protoplast, a callus, or any other population of plant cells organized into structural or functional units. The 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. The regenerable cells in the plant cells or tissue culture may be embryos, protoplasts, meristematic cells, callus tissue, pollen, leaves, anthers, roots, root tips, filaments, flowers, kernels, ears, cobs, hulls or stems. In contrast, some plant cells are not capable of regenerating to produce plants, and are referred to herein as "non-regenerable" plant cells.

The term "purified" as used herein defines that the molecule or compound is isolated in a form that is substantially free of contaminants normally associated with the molecule or compound in the natural or natural environment and means that the purity is increased as a result of separation from the other components of the original composition. The term "purified nucleic acid" is used herein to describe a nucleic acid sequence that has been separated from other compounds including, but not limited to, polypeptides, lipids, and carbohydrates.

As used herein, the term "recipient" refers to a plant or plant line that receives a trait, transgenic event, or genomic segment from a donor, and the recipient itself may or may not have the trait, transgenic event, or genomic segment in a heterozygous or homozygous state.

As used herein, the term "recurrent parent" or "recurrent plant" describes an elite line as a recipient plant line in a cross that will be used as a parent line for successive backcrosses to produce the final desired line.

As used herein, the term "recurrent parent percentage" refers to the same percentage of the progeny plants of the backcross as the recurrent parent plants used in the backcross. The percent identity to the recurrent parent may be determined experimentally by measuring genetic markers such as SNPs and/or RFLP, or may be calculated theoretically according to mathematical formulas.

As used herein, the terms "selfed," "selfing," and "selfing" refer to any process for obtaining progeny from the same plant or plant line, as well as plants produced by the process. As used herein, the term thus includes any fertilization process (wherein the ovule and pollen are both from the same plant or plant line) and the resulting plant. Generally, these terms refer to the self-pollination process and the progeny plants resulting from the self-pollination.

As used herein, the term "selecting" refers to the process of selecting a particular plant individual from a group of individuals, typically based on the particular identity, trait, characteristic, and/or molecular marker of that individual.

As used herein, the phrase "selectable marker gene excision site" refers to DNA remaining in a modified transgene locus, wherein a selectable marker gene, consisting essentially of, or a segment consisting of the original transgene locus has been deleted. The Selectable Marker Gene (SMG) excision site may thus comprise a contiguous DNA segment comprising at least 10 DNA base pairs 5 'of the SMG promoter and 10 DNA base pairs 3' of the SMG terminator.

As used herein, the phrase "transgenic element" refers to a DNA segment comprising, consisting essentially of, or consisting of: promoters, 5 'UTRs, introns, coding regions, 3' UTRs or polyadenylation signals. Polyadenylation signals include transgenic elements known as "terminators" (e.g., NOS, pinII, rbcs, hsp, tubA).

To the extent that any of the foregoing definitions are inconsistent with the definitions provided in any patent or non-patent reference incorporated by reference herein, any patent or non-patent reference cited herein, or any patent or non-patent reference found elsewhere, it should be understood that the foregoing definitions will be used herein.

Genome editing molecules may allow for the introduction of targeted genetic alterations to confer desired traits in various crop plants (Zhang et al Genome Biol [ Gene Biol ]2018;19:210; schindex et al FEBS Lett [ FEBS flash ]2018;592 (12): 1954). Desirable traits introduced into crop plants such as maize and soybean include herbicide tolerance, improved food and/or feed characteristics, male sterility, and drought stress tolerance. Nevertheless, to fully exploit the potential of genome editing methods in crop improvement, there is a need to effectively incorporate targeted genetic alterations into the germplasm of different elite crop plants adapted to different growth conditions. Such elite crop plants will also desirably comprise useful transgene loci conferring 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 improved and/or adapted for rapid incorporation of elite crop plants targeted for genetic alteration by genome editing comprising modified transgene loci, and methods of making and using such crop plants. Also provided are DNA molecules obtained from the modified transgenic loci and/or plants comprising the same, biological samples containing the DNA, nucleic acid markers suitable for detecting isolated DNA molecules, and related methods for identifying elite crop plants comprising improved and/or adapted for rapid incorporation of the modified transgenic loci targeted for genetic alteration by genome editing.

Provided herein are methods for targeted or targeted excision of a selectable marker gene or a scorable marker gene from a transgenic locus in a transgenic plant. In certain embodiments, the methods for excision of a selectable or scorable marker gene from a transgenic locus comprise targeted excision of a given selectable or scorable marker gene in a transgenic locus of a certain breeding line or crossing of a transgenic locus lacking a selectable or scorable marker gene with other plants. Other useful applications of methods for excision of selectable or scorable marker genes from transgenic loci include removal of selectable traits from certain breeding lines when it is desired to replace selectable traits in the breeding lines without disruption of other transgenic loci and/or non-transgenic loci. In certain embodiments, excision of the selectable or scorable marker gene from the transgene locus may be accompanied or followed by insertion of a new transgene conferring a substitution or other desired trait at the genomic location of the excised selectable or scorable marker gene (i.e., the excision site that remains in the genome after excision of the selectable or scorable marker gene). Transgenic plants comprising an edited genome comprising a transgenic locus in which a selectable marker gene or a scorable marker gene has been excised are also provided. In certain embodiments, the transgene locus in which the selectable marker gene has been excised does not comprise any site-specific recombinase recognition sites (e.g., lox or FRT sites). In certain embodiments, these methods result in plants, genomic DNA, biological samples, and/or DNA containing selectable marker gene excision sites, wherein the selectable marker gene, consisting essentially of, or a segment consisting of the transgene locus is deleted.

Also provided herein are methods of targeted or targeted excision (e.g., resulting in deletion) of a polynucleotide segment from a transgene locus contained in the genome of a transgenic plant, as well as the edited transgenic plant genome produced thereby and plant cells, plant parts, and plants comprising such edited plant genome. In certain embodiments, the original transgenic locus is modified by deleting a DNA segment comprising, consisting essentially of, or consisting of a DNA segment not necessary for expression of any transgene in the locus. In some cases, such non-essential DNA may be considered undesirable or even detrimental to the transgenic event and/or the function or purpose of the transgene, and thus its removal may result in a recognizable improvement in the transgenic locus and/or transgenic plant comprising such an edited genome. In certain embodiments, the removal of deleterious DNA may provide enhanced function of the modified transgene locus as compared to the absence of the deleted transgene locus. In certain embodiments, the enhanced function comprises reducing silencing of the complete transgene comprising the deleted modified transgene locus and/or increasing expression of the complete transgene comprising the deleted approved transgene locus. In addition to inserted transgenes, transgenic events generated by various methods can result in the inclusion of foreign and/or non-essential DNA sequences within the transgene locus. Non-limiting examples of non-essential DNA in a transgenic locus include synthetic cloning site sequences, replication or other duplication of the entire transgene, transgenic elements, fragments of the transgene or transgenic elements, bacterial antibiotic resistance genes (e.g., beta-lactamase (bla)), bacterial vector backbone sequences, and agrobacterium right and/or left border sequences. Plant transformation by particle bombardment can result in, among other things, replication and fragmentation of transgenic sequences. In addition to promoter sequences that drive expression of a transgene, repeated promoter sequences or fragments of promoter sequences within a transgenic locus may interfere with, obstruct or otherwise alter expression of the transgene or potentially alter expression of other genes in regions of non-essential promoter sequences. In certain embodiments, the non-essential DNA does not comprise DNA encoding a selectable marker gene, i.e., the non-essential DNA of the transgene locus and any selectable marker gene are considered separate elements for the purposes of such embodiments. In certain embodiments, the methods for excision of a segment of a transgenic locus include targeted excision of non-essential DNA, or targeted excision of non-essential DNA in conjunction with targeted excision of a selectable marker gene, e.g., in a transgenic locus of certain breeding lines. In certain embodiments, the methods for excision of a transgenic locus segment comprise crossing a plant comprising the transgenic locus modified by deletion of non-essential DNA or by deletion of non-essential DNA and a selectable marker gene with other plants. Other useful applications of methods for excision of non-essential or non-essential DNA and selectable marker genes from a transgenic locus include removal of non-essential or non-essential DNA and selectable marker genes from certain breeding lines (e.g., inbred lines). For example, it is sometimes desirable to excise or replace non-essential and/or non-essential DNA and selectable marker genes in a breeding line without disrupting other transgenic and/or non-transgenic loci. In certain embodiments, excision of non-essential DNA or excision of non-essential DNA and a selectable marker gene from a transgenic locus may be accompanied or followed by insertion of an introduced DNA sequence, such as a new transgene, that confers substitution or other desired function or trait at the location of one or more segments of the excision (i.e., the excision site remaining in the genome after excision of the deleted polynucleotide segment). Also provided is an edited transgenic plant genome comprising a transgenic locus, wherein non-essential or non-essential DNA and a selectable marker gene have been excised. Transgenic plants comprising an edited genome containing a modified transgene locus in which non-essential or non-essential DNA and a selectable marker gene have been excised are also provided. In certain embodiments, the transgene locus in which the non-essential or non-essential DNA and selectable marker gene have been excised does not comprise any site-specific recombinase recognition sites (e.g., lox or FRT sites).

The methods provided herein can be used to excise any selectable marker gene and/or non-essential DNA from a transgenic locus, wherein the DNA sequences flanking and/or comprising the selectable marker gene and/or non-essential DNA are determined or determinable. Such DNA sequences are readily identified in new transgenic events by sequencing and PCR techniques. In certain embodiments, such sequences are disclosed. Examples of transgenic loci that can be improved and used in the methods provided herein include certain maize (maize), soybean, cotton and canola transgenic loci listed in tables 1, 2, 3 and 4, respectively. DNA sequences including selectable marker genes, non-essential DNA segments and certain event regions flanking them are also depicted in the figures and provided with them.

The methods provided herein can be used to excise any selectable marker gene from a transgenic locus, including 5 'and 3' DNA sequences comprising the 5 'and 3' ends of an expression cassette comprising the selectable marker gene (e.g., a DNA segment comprising a promoter operably linked to DNA encoding a protein conferring a selectable trait, which in turn is operably linked to DNA encoding a termination or polyadenylation signal) are known or have been determined. Such 5 'and 3' DNA sequences flanking the selectable marker gene can be readily identified in new transgenic events by sequencing and PCR techniques. In certain embodiments, 5 'and 3' dna sequences flanking the selectable marker gene are disclosed. Examples of transgenic loci that can be improved and used in the methods provided herein include certain maize (maize), soybean, cotton and canola transgenic loci listed in tables 1, 2, 3 and 4, respectively. Transgenic 5 'and 3' DNA sequences flanking selectable marker genes for certain events are also depicted in the figures. Such transgenic loci listed in tables 1-4 are present in crop plants that have been cultivated, placed into commerce in some instances, and/or described in various publications by various government agencies. Databases that assemble descriptions of approved transgenic loci, including the loci listed in tables 1-4, include the International agricultural biotechnology application procurement services (International Service for the Acquisition of Agri-biotech Applications, ISAAA) database (available at the Internet site "ISAAA. Org/gmapprovadatabase/event"), the GenBit LLC database (available at the Internet site "genbitgroup. Com/en/gmo/gmodatabase"), and the biosafety information clearinghouse (Biosafety Clearing-House, BCH) database (available at the http Internet site "BCH. Cbd. Int/database/organization").

TABLE 1 maize event (transgenic loci)

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¹ Traits: IR = insect resistance; HT = herbicide tolerance; AR = antibiotic resistance; MU = mannose utilization; bf=biofuel; MS = male sterility; MSR = male sterility recovery; q = food and/or feed quality; AST = abiotic stress tolerance; YG = yield/growth.

² Each U.S. patent or patent application publication is incorporated by reference herein in its entirety.

³ The single transgene confers glyphosate tolerance to the plant and exhibits glyphosate-induced male sterility.

⁴ Resistance to coleopteran and lepidopteran pests.

TABLE 2 Soybean event (transgenic loci)

¹ Traits: ir=insectResistance; HT = herbicide tolerance; AR = antibiotic resistance; MU = mannose utilization; bf=biofuel; MS = male sterility.

³ ATCC is American type culture Collection (American Type Culture Collection), university of Massachus, 10801, va.20110 USA (for "PTA-XXXXX" collections).

⁴ NCIMB is national industry, food and marine bacterial collection (National Collection of Industrial, food and Marine Bacteria), frapperson building (Ferguson Building), claibuten manor (Craibstone Estate), bucksburn, abberan AB9YA (Aberdeen AB9 YA), scotland.

⁵ HT for the following: 2,4-D; glyphosate and glufosinate; also known as pdab8264.44.06.1.

⁶ Independent IR/HT and HT events were combined by breeding. The IR/HT events (Cry 1F, cry1Ac synpro (Cry 1 Ac) and PAT) are DAS81419-2 deposited as PTA-12006 with the ATCC, also known as DAS81419-2.

⁷ Elk Mound Seed (Elk ground Seed), railroad street number 308, elk Mound, USA 54739, wisconsin.

⁸ HT for dicamba.

⁹ HT for glyphosate and clomazone herbicides.

¹⁰ HT against glufosinate-ammonium and mesotrione herbicides.

TABLE 3 Cotton event (transgenic loci)

¹ Traits: IR = insect resistance; HT = herbicide tolerance; AR = antibiotic resistance; sm=screenable marker.

² cry1Ac cotton event 3006-210-23 and cry1F cotton event 281-24-236 is described in US 7,179,965; seeds containing two events deposited as PTA-6233 at the ATCC.

³ Comprising both MON531 chimeric Cry1A and MON15985X Cry2Ab insertions.

⁴ Tolerance to dicamba and glufosinate herbicides.

TABLE 4 canola event (transgenic loci)

¹ Traits: HT = herbicide tolerance; MS = male sterility

The sequences of certain transgene loci are listed in tables 1-4 (e.g., SEQ ID NOS: 1-34), the patent references listed therein and incorporated by reference herein, and elsewhere in the present disclosure. Such sequences include 5 'and 3' DNA sequences flanking the selectable marker gene, non-essential DNA sequences, selectable marker gene cassette sequences, and other expression cassette sequences conferring useful traits (e.g., herbicide tolerance, insect resistance, biofuel use). Alleles or other variant sequences corresponding to the sequences listed in tables 1-4 and elsewhere in the disclosure that may be present in certain variant transgenic plant loci can also be improved by: sequences in variants (which correspond to tables 1-4 (e.g., SEQ ID NOS: 1-34), the sequences described therein and elsewhere in the patent references and the disclosure herein incorporated by reference) are identified by making an alignment (e.g., using CLUSTAL O1.2.4 with default parameters) and making corresponding changes in the allelic or other variant sequences. Such allele or other variant sequences include sequences having at least 85%, 90%, 95%, 98%, or 99% sequence identity across all or at least 20, 40, 100, or 500, 1,000, 2,000, 4,000, 8,000, 10,000, or 12,000 nucleotides of tables 1-4 (e.g., SEQ ID NOS: 1-34), the patent references listed therein and incorporated by reference herein, and the sequences listed elsewhere in the present disclosure. Also provided are plants, genomic DNA and/or isolated DNA obtained from plants listed in tables 1-4 comprising modifications to a transgene locus thereof comprising a selectable marker gene excision site, wherein a selectable marker gene comprising, consisting essentially of or a segment consisting of the transgene locus is deleted. Also provided herein are plants, genomic DNA, and/or isolated DNA obtained from plants listed in tables 1-4, comprising modifications to their transgenic loci that enhance the function of the transgenic loci, including the deletion of non-essential DNA from the transgenic loci. In certain embodiments, the functional enhancement modification can comprise a deletion of a segment comprising, consisting essentially of, or consisting of: replication of the transgene; replication of the transgenic element; and/or a fragment of the transgene; optionally, wherein the replication and/or fragment of the transgenic element is a promoter or a replication and/or fragment of a polyadenylation signal.

The modified transgene loci provided herein can be used in a variety of breeding protocols to obtain or use elite crop plants comprising the modified transgene loci and targeted genetic alterations in some ways. Such elite crop plants may be inbred plant lines or may be hybrid plant lines. In certain embodiments, one or more modified transgenic loci (e.g., the transgenic loci in tables 1-4 that have been subjected to genome editing) are introgressed into a desired donor system comprising elite crop plant germplasm, and then optionally subjected to a genome editing molecule to obtain a plant comprising the modified transgenic locus and a targeted genetic alteration introduced by the genome editing molecule. Introgression may be achieved by backcrossing a plant comprising the modified transgene locus with a recurrent parent comprising the desired elite germplasm and selecting for progeny having the modified transgene locus and the recurrent parent germplasm. Such backcrosses can be repeated and/or complemented by molecular assisted breeding techniques using SNPs or other nucleic acid markers to select recurrent parent germplasm until a desired recurrent parent percentage (e.g., at least 95%, 96%, 97%, 98%, or 99%) is obtained. FIG. 11 (bottom "alternative" panel) shows a non-limiting illustrative depiction of a protocol for obtaining plants with both modified transgene loci and targeted genetic alterations, wherein one or more modified transgene loci ("events" in FIG. 11) are present in line A and then transferred into elite crop plant germplasm by introgression. In the non-limiting illustration of FIG. 11, introgression may be achieved by crossing "line A" comprising one or more modified transgene loci with elite germplasm, and then backcrossing the progeny of the cross comprising the modified transgene locus with elite germplasm as recurrent parent to obtain a "universal donor" (e.g., line A+ in FIG. 11) comprising one or more modified transgene loci. Such elite germplasm containing modified transgene loci (e.g., the "universal donor" of fig. 11) can then be subjected to genome editing molecules that introduce other targeted genetic alterations in the genome of the elite crop plant containing the modified transgene loci. In certain embodiments in which more than one modified transgene locus is present in a elite crop plant (e.g., the "universal donor" of fig. 11), the modified transgene locus (the "event" in fig. 11) can be removed to obtain a elite crop plant with a subset of modified transgene loci and targeted genetic alterations. In certain embodiments, it is also desirable to expand the population of inbred elite crop plants or seeds thereof comprising the modified transgene locus by selfing. Inbred progeny of such inbred plants can be used for commercial sale as such, in which case the crop can grow a variety of non-hybrid crops (e.g., typically but not always in soybean, cotton or canola). In certain embodiments, inbred progeny of the inbred plant can be used as a pollen donor or acceptor for hybrid seed production (e.g., most commonly found in maize, but also in cotton, soybean, and canola).

Also provided herein are hybrid plant lines comprising elite crop plant germplasm, a modified transgene locus, and in some aspects additional targeted genetic alterations. Methods for producing such hybrid seed may include crossing elite crop plant lines, wherein at least one of the pollen donor or acceptor comprises at least a modified transgene locus and/or an additional targeted genetic alteration. In certain embodiments, the pollen donor and recipient will comprise germplasm of different heterosis populations and provide hybrid seeds and plants that exhibit heterosis. In certain embodiments, the pollen donor and recipient can each comprise a different modified transgene locus that confers a different trait (e.g., herbicide tolerance or insect resistance), a different type of trait (e.g., tolerance to a different herbicide or a different insect such as coleopteran or lepidopteran insect), or a different mode of action of the same trait (e.g., resistance to coleopteran by two different modes of action or resistance to lepidopteran insect by two different modes of action). In certain embodiments, pollen receptors will be rendered male sterile or conditionally male sterile. Methods for inducing male sterility or conditional male sterility include castration (e.g., emasculation), cytoplasmic male sterility, chemical crossing agents or systems, transgenes or transgene systems, and/or one or more mutations in one or more endogenous plant genes. Descriptions of various male sterility systems that may be applicable to elite crop plants provided herein are in Wan et al Molecular Plant; 12,3, (2019): 321-342 and US 8,618,358; US 20130031674; and US 2003188347. In certain embodiments, it is desirable to use genome editing molecules to effect modification of a transgene locus and/or targeted genetic alteration in elite crop plants or other germplasm. Techniques for achieving genome editing in crop plants (e.g., corn) include the use of morphogenic factors, such as Wuschel (WUS), ovule Development Proteins (ODPs), and/or babybom (BBMs), which can increase the efficiency of restoring plants with desired genome editing. In some aspects, the morphogenic factors include WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, WOX9, BBM2, BMN3, and/or ODP2. In certain embodiments, compositions and methods using WUS, BBM, and/or ODP, as well as other techniques that may be suitable for achieving genome editing in elite crop plants and other germplasm, are set forth in US 20030082813, US 20080134353, US 20090328252, US 20100100981, US 20110165679, US 20140157453, US 20140173775, and US 20170240911, each of which is incorporated by reference in its entirety. In certain embodiments, genome editing may be accomplished in a regenerable plant part of a elite crop plant (e.g., plant embryo) by transiently providing a gene editing molecule or polynucleotide encoding the same, and it is not necessary to incorporate a selectable marker gene into the plant genome (e.g., U.S. Pat. No. 3,979 and U.S. Pat. No. 3, 20180273960, both of which are incorporated herein by reference in their entireties; svitashev et al Nat Commun [ Nature communication ].2016, 7:13274).

Provided herein are modified forms of an approved transgenic locus, the unmodified form of the approved transgenic locus (in certain embodiments, "unmodified form" is "original form", "original transgenic locus", etc.) comprising at least one selectable marker gene. In a modified form, at least one selectable marker has been deleted from an unmodified approved transgene locus with a genome editing molecule as described elsewhere herein. In certain embodiments, targeted genome editing and deletion of the selectable marker gene does not affect any other function of the approved transgene locus. In certain embodiments, the deleted selectable marker gene confers resistance to antibiotics, resistance to herbicides, or the ability to grow on a specific carbon source such as mannose. In certain embodiments, the selectable marker gene comprises DNA encoding: phosphinothricin Acetyl Transferase (PAT), glyphosate tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosate Oxidase (GOX), neomycin phosphotransferase (npt), hygromycin phosphotransferase (hyg), aminoglycoside adenyltransferase or phosphomannose isomerase (pmi). In certain embodiments, the modified locus does not comprise a site-specific recombination system DNA recognition site, e.g., in certain embodiments, the modified locus does not comprise a lox or FRT site. In certain embodiments, the selectable marker gene to be deleted is flanked by Protospacer Adjacent Motif (PAM) sites operably linked in the unmodified version of the approved transgene locus. Thus, in certain embodiments of the modified locus, the PAM site flanks the excision site of the deleted selectable marker gene. In certain embodiments, P The AM site is recognized by an RNA-dependent DNA endonuclease (RdDe); for example, type II or type V RdDe. In certain embodiments, the deleted selectable marker gene is replaced with the introduced DNA sequence in the modified approved transgene locus, as discussed in further detail elsewhere herein. For example, in certain embodiments, the introduced DNA sequence comprises a trait expression cassette, such as a trait expression cassette of another transgenic locus. In addition to the deletion of the selectable marker gene, in certain embodiments, at least one copy of the repeat sequence is deleted from the unmodified, approved transgene locus with a genome editing molecule. In certain embodiments, the deletion of the repeat sequence enhances the function of the modified approved transgene locus. In certain embodiments, the approved modified transgene locus is: (i) Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017, MON89034, MIR162, MIR604, NK603, SYN-E3272-5, 5307, DAS-40278, DP-32138, DP-33121, HCEM485, LY038, MON863, MON87403, MON87419, MON87460, MZIG 0JG, MZIR098, MIR604, NK603, SYN-E3272-5, 5307, DAS-40278, DP-32138, DP-33121, HCEM485, LY038, MON863, MON87403, MON87460, MZIG 0JG, MZIR098,

98140. And/or the TC1507 transgene locus; (ii) A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788, and transgenic soybean plant genome>

And/or a SYHT0H2 transgene locus; (iii) DAS-21023-5, DAS-24236-5, COT102, LL cotton25 (LLcotton 25), MON15985, MON88701, and/or MON88913 transgenic loci in the genome of the transgenic cotton plant; or (iv) the GT73, HCN28, MON88302, and/or MS8 transgene locus in the genome of a transgenic canola plant. Also provided herein are plants comprising any of the above modified transgenic loci.

Excision of the selectable marker gene and/or non-essential DNA can be accomplished by use of suitable gene editing molecules that can introduce blunt-ended or staggered double-stranded DNA breaks in the 5 'and 3' DNA sequences flanking or containing the selectable marker gene and/or non-essential DNA of the transgene locus. Such blunt-ended or staggered dsDNA breaks may be introduced in or adjacent to promoters and terminators or polyadenylation signals of selectable marker genes. Typically, the break is introduced at or just 5 'to the DNA comprising the promoter and at or just 3' to the DNA comprising the terminator or polyadenylation signal. However, such breaks may also be introduced in the DNA comprising the promoter and terminator of the selectable marker gene or the polyadenylation signal. In certain embodiments, the gene editing molecule may comprise zinc finger nucleases, zinc finger nicking enzymes, TALENs, and/or TALE nicking enzymes that introduce double strand breaks in DNA segments flanking the sequence to be deleted from the genome (e.g., selectable marker gene cassettes and/or non-essential DNA). In certain embodiments, the gene editing molecule comprises RdDe and guide RNA directed to a DNA target comprising pre-existing PAM sites in DNA flanking or comprising a promoter and terminator for a selectable marker gene in the genome of the transgenic plant. Such PAM sites can be recognized by RdDe and appropriate guide RNAs (which are directed to DNA sequences adjacent to PAM) to provide cleavage within or near the DNA site targeted for cleavage. In certain embodiments, PAMs are recognized by the same class and/or type of RdDe (e.g., class II or class V type 2) or the same RdDe (e.g., both PAMs are recognized by the same Cas9 or Cas 12 RdDe). By using pre-existing PAM sites (e.g., within or adjacent to DNA segments flanking the selectable marker gene cassette and/or non-essential DNA), the guide RNA can be directed to the DNA sites targeted for cleavage. Non-limiting examples of such pre-existing PAM sites present in polynucleotides that may be used by suitable guide RNAs to guide RdDe or RNA-dependent nicking enzymes in DNA segments flanking selectable marker gene cassettes of certain transgene loci are listed in tables 5, 6, 7, 8 and 9 of the examples. In certain embodiments, selectable marker genes conferring herbicide tolerance or antibiotic resistance are excised from a transgenic locus having a primary function conferring insect resistance, male sterility, or biofuel use. In certain embodiments, the selectable marker gene conferring antibiotic resistance is excised from a transgenic locus having a primary function of conferring herbicide tolerance.

In certain embodiments, the edited transgenic plant genomes, transgenic plant cells, parts or plants containing these genomes, and DNA molecules obtained therefrom may lack one or more non-essential DNA and/or selectable and/or scorable markers found in the original event (transgenic locus) and comprise selectable marker gene excision sites or scorable marker gene excision sites. When a segment comprising the Selectable Marker Gene (SMG) of the original transgene locus has been deleted, the selectable marker gene excision site may comprise a contiguous segment of DNA comprising at least 10 DNA base pairs 5 'of the SMG promoter and 10 DNA base pairs 3' of the SMG terminator, wherein the entire selectable marker gene (e.g., an expression cassette in the original transgene locus comprising a promoter operably linked to DNA encoding a selectable marker protein operably linked to a terminator) has been deleted. In certain embodiments in which a segment of the selectable marker gene comprising the original transgene locus has been deleted, the selectable marker gene excision site can comprise a contiguous segment of DNA comprising at least 10 DNA base pairs 5 'to the excision site and 10 DNA base pairs 3' to the excision site, wherein the entire selectable marker gene (e.g., an expression cassette in the original transgene locus comprising a promoter operably linked to DNA encoding a selectable marker protein operably linked to a polyadenylation sequence) and DNA in the original transgene locus located 5 'to the SMG promoter and/or 3' to the SMG polyadenylation signal has been deleted. In such embodiments in which DNA comprising a selectable or scorable marker gene is deleted, the selectable marker excision site may comprise DNA of at least 10 base pairs 5 'to the excision site and DNA of 10 base pairs 3' to the excision site (e.g., DNA 5 'to the SMG promoter and/or 3' to the SMG polyadenylation signal prior to deletion of the fragment), wherein all of the selectable marker gene sequence is absent and all or less than all of the DNA flanking the selectable or scorable marker gene sequence is present. In any of the above embodiments or other embodiments, the contiguous DNA segment comprising the selectable marker gene excision site can further comprise an insertion of 1 to about 2, 5, 10, 20 or more nucleotides between the DNA located 5 'and 3' of the excision site. Such insertions may be caused by endogenous DNA repair and/or recombination events at double strand breaks introduced at the excision site and/or by intentional insertion of oligonucleotides. In certain embodiments in which a segment consisting essentially of the selectable marker gene of the original transgene locus has been deleted, the selectable marker gene excision site may be a contiguous segment having at least 10 DNA base pairs 5 'of the excision site and 10 DNA base pairs 3' of the excision site, wherein less than the entire selectable marker gene (e.g., an expression cassette in the original transgene locus comprising a promoter operably linked to DNA encoding a selectable marker protein operably linked to a polyadenylation signal sequence) has been deleted. In certain of the foregoing embodiments in which a segment consisting essentially of the selectable marker gene of the original transgene locus has been deleted, the selectable marker excision site may thus comprise 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 5 'end and/or the 3' end of the selectable marker gene cassette (e.g., DNA comprising the selectable marker gene cassette promoter and/or polyadenylation signal fragment). In certain embodiments in which a segment consisting of the selectable marker gene of the original transgene locus has been deleted, the selectable marker gene excision site may comprise a contiguous segment of DNA comprising at least 10 DNA base pairs 5 'of the excision site and 10 DNA base pairs 3' of the excision site, wherein the entire selectable marker gene (e.g., an expression cassette in the original transgene locus comprising a promoter operably linked to DNA encoding the selectable marker protein operably linked to a polyadenylation signal sequence) has been deleted. In such embodiments where DNA consisting of the selectable marker gene is deleted, the selectable marker excision site can comprise at least 10 base pairs of DNA 5 'to the excision site and 10 base pairs of DNA 3' to the excision site, wherein all of the selectable marker gene sequence is absent and all of the DNA flanking the selectable marker sequence is present. Deletions from the transgene locus that comprise, consist essentially of, or consist of DNA segments that provide a scorable marker gene excision site having characteristics similar to those of the selectable marker gene excision site described above. The original transgene locus (event), including those described in tables 1-4 and depicted in the figures, may contain selectable transgene markers that confer herbicide tolerance, antibiotic resistance, or the ability to grow on a carbon source. Selectable marker transgenes that may confer herbicide tolerance include genes encoding Phosphinothricin Acetyltransferase (PAT), glyphosate tolerant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS), and Glyphosate Oxidase (GOX). Selectable marker transgenes that can confer antibiotic resistance include genes encoding neomycin phosphotransferase (npt), hygromycin phosphotransferase, and aminoglycoside adenyltransferase. Transgenes encoding phosphomannose isomerase (pmi) may confer the ability to grow on mannose. The original transgene locus (event), including certain events listed in tables 1-4, may contain scorable transgene markers that can be detected by enzymatic, histochemical, nucleic acid detection (e.g., sequencing, amplification, hybridization, SNP) or other assays. The scorable marker gene may include a gene encoding β -glucuronidase (uid) or a fluorescent protein (e.g., GFP, RFP, or YFP). Such selectable or scorable marker transgenes can be excised from the original transgene locus by: the transgenic loci are contacted with one or more gene editing molecules (e.g., rdDe and guide RNAs that are directed to PAM sites located at the 5 'and 3' ends of expression cassettes comprising selectable marker transgenes) that introduce double strand breaks in the transgenic loci at the 5 'and 3' ends of the expression cassettes comprising selectable marker transgenes, and plant cells, plant parts, or plants in which the selectable or scorable markers have been wholly or partially excised are selected. Provided herein are plants, edited plant genomes, biological samples, and DNA molecules (e.g., including isolated or purified DNA molecules) comprising selectable marker gene excision sites. Also provided herein are nucleic acid markers suitable for detecting selectable marker gene excision sites and/or scorable marker gene excision sites and methods for detecting the presence of a DNA molecule comprising a selectable marker excision site and/or scorable marker gene excision site.

Also provided herein are methods and reagents (e.g., nucleic acid markers including nucleic acid probes and/or primers) for detecting plant, edited plant genomes and biological samples containing DNA molecules comprising selectable marker gene excision sites and/or non-essential DNA deletions. Detection of DNA molecules can be achieved by any combination of nucleic acid amplification (e.g., PCR amplification), hybridization, sequencing, and/or mass spectrometry-based techniques. The methods described in US 20190136331 and US 9,738,904 for detecting a linked nucleic acid in an unmodified transgenic locus may be suitable for detecting a nucleic acid provided herein, both of which are incorporated herein by reference in their entirety. In certain embodiments, such detection is accomplished by amplification and/or hybridization-based detection methods that use methods (e.g., selective amplification primers) and/or probes (e.g., capable of selectively hybridizing or producing specific primer extension products) that specifically recognize the target DNA molecule (e.g., selectable marker gene excision site) but do not recognize DNA from the unmodified transgene locus. In certain embodiments, the hybridization probes can comprise a detectable label (e.g., fluorescent label, radiolabel, epitope label, and chemiluminescent label). In certain embodiments, single nucleotide polymorphism detection assays may be suitable for detecting a target DNA molecule (e.g., selectable marker gene excision sites).

In certain embodiments, selectable or scorable marker transgenes may be inactivated. Inactivation may be achieved by modification, including insertion, deletion and/or substitution of one or more nucleotides in the promoter element, 5' or 3' untranslated region (UTR), intron, coding region and/or 3' terminator and/or polyadenylation signal of the selectable marker transgene. Such modifications may inactivate selectable or scorable marker transgenes by eliminating or reducing promoter activity, introducing missense mutations, and/or introducing premature stop codons. In certain embodiments, selectable and/or scorable marker transgenes may be replaced by introduced transgenes. In certain embodiments, the original transgene locus contacted with a gene editing molecule that introduces double strand breaks in the transgene loci at the 5 'and 3' ends of the selectable marker and/or scorable transgene expression cassette may also be contacted with a suitable donor DNA template comprising an expression cassette flanked by DNA homologous to the remaining DNA in the transgene locus located 5 'and 3' of the selectable marker excision site. In certain embodiments, the coding region of the selectable and/or scorable marker transgene may be replaced with another coding region such that the replacement coding region is operably linked to the promoter and 3' terminator or polyadenylation signal of the selectable and/or scorable marker transgene.

In certain embodiments, the edited transgenic plant genomes provided herein may comprise additional newly introduced transgenic-comprising DNA sequences (e.g., expression cassettes) inserted into the transgenic loci of a given event. The introduced transgene inserted at the transgene locus of the event after the initial isolation of the event can be obtained by: inducing a double strand break at a site within the original transgene locus (e.g., with a genome editing molecule comprising RdDe and one or more suitable guide RNAs); suitable engineered zinc finger nucleases; TALEN proteins, etc.) and provides exogenous transgenes in a donor DNA template that can be integrated at the double strand break site (e.g., by Homology Directed Repair (HDR) or non-homologous end joining (NHEJ)). In certain embodiments, the introduced transgene may be integrated at a selectable marker gene excision site created by: a pre-existing PAM site in the DNA segment flanking or comprising the 5 'end or 3' end of the selectable marker gene is used with a suitable RdDe, guide RNA. In certain embodiments, such deletions and substitutions are accomplished by: introducing a dsDNA break in a DNA segment flanking or comprising the 5 'end or 3' end of a selectable marker gene and providing a new expression cassette on a donor DNA template or other DNA template suitable for integration by NHEJ or MMEJ (micro homology mediated end ligation). Suitable expression cassettes for insertion include DNA molecules comprising a promoter operably linked to DNA and/or RNA molecules encoding a protein conferring a useful trait, which in turn is operably linked to a polyadenylation signal or terminator element. In certain embodiments, such expression cassettes may also comprise a 5'utr, a 3' utr, and/or an intron. 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 composition, protein content, starch content, etc.). Expression cassettes suitable for insertion include those listed in table 1 or contained in any event (transgene locus) set forth in the figures, which confers insect resistance, herbicide tolerance, biofuel use, male sterility or other useful traits.

In certain embodiments, the plants provided herein (including plants having one or more modified transgene loci comprising a selectable marker gene excision site and/or a deletion of one or more non-essential DNA) may further comprise one or more targeted genetic alterations introduced by one or more gene editing molecules or systems. Also provided are methods of introducing targeted genetic alterations into plants, including plants having one or more modified transgene loci comprising selectable marker gene excision sites and/or deletions of one or more non-essential DNA. Such targeted genetic alterations include conferring, as compared to a control plant lacking the targeted genetic alteration, increased yield, increased food and/or feed characteristics (e.g., increased oil, starch, protein, or amino acid quality or quantity), increased nitrogen utilization efficiency, increased biofuel utilization characteristics (e.g., increased ethanol yield), male sterility/conditional male sterility systems (e.g., by targeting endogenous MS26, MS45, and MSCA1 genes), herbicide tolerance (e.g., by targeting endogenous ALS, EPSPS, HPPD or other herbicide target genes), delayed flowering, non-flowering, increased biotic stress resistance (e.g., resistance to insect, nematode, bacterial, or fungal damage), increased abiotic stress resistance (e.g., resistance to drought, cold, heat, metal, or salt), increased lodging resistance, increased growth rate, increased biomass, increased tillering, increased branching, delayed flowering time, delayed senescence, increased flowering, improved architecture for high density planting, improved photosynthesis, increased root mass, increased cell division, improved vigor of the targeted seedlings, greatly altered metabolic rates, small cell viability, and those of the targeted seedlings. Types of targeted genetic alterations that can be introduced include insertions, deletions, and substitutions of one or more nucleotides in the genome of a crop plant. Sites in endogenous plant genes for targeting genetic alterations include promoters, coding and non-coding regions (e.g., 5'utr, introns, splice donor and acceptor sites, and 3' utr). In certain embodiments, the targeted genetic alteration comprises insertion of regulatory or other DNA sequences in the endogenous plant gene. Non-limiting examples of regulatory sequences that can be inserted into endogenous plant genes with gene editing molecules to achieve targeted genetic alterations that confer useful phenotypes include those listed in U.S. patent application publication 20190352655 (which is incorporated herein by example), such as: (a) an auxin response element (AuxRE) sequence; (b) At least one D1-4 sequence (Ulmasov et al (1997) Plant Cell [ Plant Cell ], 9:1963-1971), (c) at least one DR5 sequence (Ulmasov et al (1997) Plant Cell [ Plant Cell ], 9:1963-1971); (d) At least one m5-DR5 sequence (Ulmasov et al (1997) Plant Cell [ Plant Cell ], 9:1963-1971); (e) at least one P3 sequence; (f) Small RNA recognition site sequences bound by the corresponding small RNAs (e.g., siRNA, micrornas (mirnas), trans-acting siRNA as described in us patent No. 8,030,473, or staged sRNA as described in us patent No. 8,404,928; both of these cited patents are incorporated herein by reference); (g) a microrna (miRNA) recognition site sequence; (h) The sequence recognizable by the specific binding agent comprises a microrna (miRNA) recognition sequence of an engineered miRNA, wherein the specific binding agent is the corresponding engineered mature miRNA; (i) a transposon recognition sequence; (j) Sequences recognized by an ethylene response element binding factor-associated amphipathic repression (EAR) motif; (k) Splice site sequences (e.g., donor site, branching site, or acceptor site; see, e.g., splice sites and splice signals listed in the internet site lemur [ dot ] amu [ dot ] edu [ dot ] pl/share/ERISdb/home. Html); (l) A recombinase recognition site sequence recognized by a site-specific recombinase; (m) a sequence encoding an RNA or amino acid aptamer or RNA riboswitch, the specific binding agent is the corresponding ligand, and the change in expression is up-or down-regulation; (n) a hormone-responsive element recognized by a nuclear receptor or hormone-binding domain thereof; (o) a transcription factor binding sequence; and (p) a multi-comb response element (see Xiao et al (2017) Nature Genetics [ Nature Genetics ],49:1546-1552, doi:10.1038/ng.3937). Non-limiting examples of target maize genes that can be targeted gene editing to confer useful traits include: (a) ZmIPK1 (herbicide and phytate-reduced corn; shukla et al Nature 2009; 459:437-41); (b) ZmGL2 (reduction of epidermal wax in leaves; char et al Plant Biotechnol J [ journal of plant biotechnology ]2015; 13:1002); (c) ZmMTL (induction of haploid plants; kelliher et al Nature: 2017; 542:105); (d) Wx1 (high amylopectin content; US 20190032070; incorporated herein by reference in its entirety); (e) TMS5 (thermo-sensitive male sterility; li et al J Genet Genomics. [ genetics ]2017; 44:465-8); (f) ALS (herbicide tolerance; svitashev et al; plant Physiol. [ Physiol. ]2015; 169:931-45); and (g) ARGOS8 (drought stress tolerance; shi et al, plant Biotechnol J. [ J.plant Biotechnology ]2017; 15:207-16). Non-limiting examples of target soybean genes that can be targeted gene editing to impart useful traits include: (a) FAD2-1A, FAD2-1B (increased oleic acid content; haun et al; plant Biotechnol J. [ J. Plant Biotechnology ]2014; 12:934-40); (b) FAD2-1A, FAD2-1B, FAD A (increased oleic acid and decreased linolenic acid content; demorest et al, BMC Plant Biol [ BMC Plant Biol ]2016; 16:225); and (c) ALS (herbicide tolerance; svitashev et al; plant Physiol. [ Plant Physiol. ]2015; 169:931-45). Non-limiting examples of target brassica genes that can be targeted gene editing to confer useful traits include: (a) FRIGIDA gene conferring early flowering (Sun Z et al J Integr Plant Biol [ Proprietary ]2013; 55:1092-103); and (b) ALS (herbicide tolerance; US 20160138040, incorporated herein by reference in its entirety). Non-limiting examples of target genes in crop plants (including corn and soybean) that can undergo targeted genetic alterations that confer useful phenotypes include those listed in U.S. patent application nos. 20190352655, 20200199609, 20200157554, and 20200231982, each of which is incorporated herein in its entirety; and Zhang et al (Genome Biol. [ Gene Biol ]2018; 19:210).

Gene editing molecules useful in the methods provided herein include molecules capable of introducing double-strand breaks ("DSB") or single-strand breaks ("SSB") in double-stranded DNA, such as in genomic DNA or target genes located within genomic DNA, or accompanying guide RNA or donor or DNA template polynucleotides. Examples of such gene editing molecules include: (a) Nucleases, including RNA-guided nucleases, RNA-guided DNA endonucleases or RNA-guided DNA endonucleases (RdDe), class 1 CRISPR-type nuclease systems, class II Cas nucleases, cas9, nCas9 nickases, class 2V Cas nucleases, cas12a nucleases, nCas12a nickases, cas12d (CasY), cas12e (CasX), cas12b (C2C 1), cas12C (C2C 3), cas12i, cas12j, cas14, engineered nucleases, codon optimized nucleases, zinc Finger Nucleases (ZFNs) or nickases, transcription activator-like effector nucleases (TAL-effector nucleases or TALENs) or nickases (TALE-nickases), argonaute and meganucleases or engineered meganucleases; (b) Polynucleotides encoding one or more nucleases capable of effecting a site-specific change in a target nucleotide sequence (including the introduction of a DSB or SSB); (c) Guide RNA (gRNA) for RNA-guided nucleases, or DNA encoding gRNA for RNA-guided nucleases; (d) a donor DNA template polynucleotide; and (e) other DNA templates (dsDNA, ssDNA, or combinations thereof) suitable for insertion at breaks in genomic DNA, e.g., by non-homologous end joining (NHEJ) or micro-homology mediated end joining (MMEJ).

CRISPR-type genome editing can be applied to the plant cells and methods provided herein in several ways. CRISPR elements, such as gene editing molecules comprising a CRISPR endonuclease and a CRISPR guide RNA (including single guide RNA or guide RNA combined with tracrRNA or scoutna, or polynucleotides encoding the same), can be used to effect genome editing without the presence of residues of the CRISPR element or selectable genetic markers in the offspring. In certain embodiments, CRISPR elements are provided directly to eukaryotic cells (e.g., plant cells), systems, methods, and compositions as isolated molecules, as isolated or semi-purified products of a cell-free synthesis process (e.g., in vitro translation), or as isolated or semi-purified products of a cell-based synthesis process (e.g., as in bacterial or other cell lysates). In certain embodiments, the genome inserted CRISPR elements can be used in plant lines suitable for use in the methods provided herein. In certain embodiments, plants or plant cells used in the systems, methods, and compositions provided herein can comprise transgenes that express CRISPR endonucleases (e.g., cas9, cpf1 type, or other CRISPR endonucleases). In certain embodiments, one or more CRISPR endonucleases with unique PAM recognition sites can be used. The guide RNAs (sgrnas or crrnas and tracrrnas) form RNA-directed endonuclease/guide RNA complexes that can specifically bind to sequences in the gDNA target site that are adjacent to the Protospacer Adjacent Motif (PAM) sequence. The type of RNA-guided endonuclease typically informs of the location of the appropriate PAM site and the design of the crRNA or sgRNA. G-rich PAM sites, e.g., 5' -NGG, are typically targeted for design of crRNA or sgRNA for use with Cas9 proteins. Examples of PAM sequences include 5'-NGG (streptococcus pyogenes (Streptococcus pyogenes)), 5' -ngagaa (streptococcus thermophilus (Streptococcus thermophilus) CRISPR 1), 5'-NGGNG (streptococcus thermophilus CRISPR 3), 5' -NNGRRT or 5'-NNGRR (staphylococcus aureus (Staphylococcus aureus) Cas9, saCas 9), and 5' -NNNGATT (neisseria meningitidis (Neisseria meningitidis)). The T-rich PAM site (e.g., 5'-TTN or 5' -TTTV, where "V" is A, C or G) is typically targeted for the design of crRNA or sgRNA for use with Cas12a proteins. In some cases, cas12a may also recognize the 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 (where N is defined as any nucleotide). Cpf1 endonuclease and corresponding guide RNA and PAM sites are disclosed in U.S. patent application publication 2016/0208243A1, the disclosure of which is incorporated herein by reference for DNA encoding Cpf1 endonuclease and guide RNA and PAM sites. The Cpf 1-based editing system may or may not contain tracrRNA. One or more of a wide variety of CRISPR guide RNAs that interact with CRISPR endonucleases integrated into the plant genome or otherwise provided to a plant can be used for gene editing for providing a desired phenotype or trait, trait screening, or gene editing-mediated trait introgression (e.g., for introducing a trait into a new genotype without backcrossing with or with limited backcrossing with a recurrent parent). Multiple endonucleases can be provided in an expression cassette with appropriate promoters to allow editing of multiple genomic sites.

CRISPR techniques for editing genes of eukaryotes are disclosed in U.S. patent application publication 2016/013008 A1 and US 2015/0344912 A1 and U.S. patent nos. 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. Cpf1 endonuclease and corresponding guide RNA and PAM sites are disclosed in U.S. patent application publication 2016/0208243A 1. Other CRISPR nucleases that can be used to edit the genome include Cas12b and Cas12c (see Shmakov et al (2015) mol. Cell [ Molecular cells ],60:385-397; harrington et al (2020) Molecular Cell [ Molecular cells ] doi:10.1016/j. Molcel 2020.06.022) and CasX and CasY (see Burstein et al (2016) Nature [ Nature ], doi: 10.1038/natural 21059; harrington et al (2020) Molecular Cell [ Molecular cells ] doi:10.1016/j. Molcel.2020.06.022), or Cas12j (Pausch et al, (2020) Science [ sciences ]10.1126/science.abb 1400). Plant RNA promoters for expression of CRISPR guide RNA and plant codon optimized CRISPR Cas9 endonucleases are disclosed in International patent application PCT/US 2015/018104 (published as WO 2015/131101 and claiming priority to U.S. provisional patent application 61/945,700). Methods of genome editing using CRISPR techniques in plants are disclosed in U.S. patent application publications US 2015/0082478 A1 and US 2015/0059010 A1 and international patent application PCT/US 2015/038767 A1 (published as WO 2016/007147 and claiming priority from U.S. provisional patent application 62/023,246). All patent publications cited in this paragraph are incorporated herein by reference in their entirety. In certain embodiments, RNA-guided endonucleases are used that leave blunt ends after cleavage of the target site. Blunt-end cutting RNA-guided endonucleases include Cas9, cas12c, and Cas12 h (Yan et al, 2019). In certain embodiments, RNA-guided endonucleases are used that leave staggered single-stranded DNA overhangs after cleavage of the target site. The misend-cleaving RNA-guided endonucleases include Cas12a, cas12b, and Cas12e.

These methods may also use sequence-specific endonucleases or sequence-specific endonucleases and guide RNAs that cleave single DNA strands in dsDNA target sites. This cleavage of a single DNA strand in a dsDNA target site is also referred to herein and elsewhere as "nicking" and can be accomplished by various "nicking enzymes" or systems that provide nicking. Nicking enzymes that may be used include nCas9 (Cas 9 comprising a D10A amino acid substitution), nCas12a (e.g., cas12a comprising an R1226A amino acid substitution; yamano et al, 2016), cas12i (Yan et al 2019), zinc finger nicking enzymes (e.g., as disclosed in Kim et al 2012), TALE nicking enzymes (e.g., as disclosed in Wu et al, 2014), or combinations thereof. In certain embodiments, the system providing nicks can comprise a Cas nuclease (e.g., cas9 and/or Cas12 a) and a guide RNA molecule with at least one base mismatch to the DNA sequence in the target editing site (Fu et al, 2019). In certain embodiments, genomic modifications may be introduced to a target editing site by creating single strand breaks (i.e., "gaps") at genomic positions no more than about 10, 20, 30, 40, 50, 60, 80, 100, 150, or 200 DNA base pairs apart. In certain illustrative and non-limiting embodiments, two nicking enzymes (i.e., CAS nucleases introducing single-stranded DNA breaks, including nCas9, nCas12a, CAS12i, zinc finger nicking enzymes, TALE nicking enzymes, combinations thereof, etc.) or nicking enzyme systems can direct sites in the vicinity of a cut that are no more than about 10, 20, 30, 40, 50, 60, 80, or 100 DNA base pairs apart. Where RNA-guided nicking enzymes and RNA guides are used, the RNA guides are adjacent to PAM sequences that are sufficiently close (i.e., no more than about 10, 20, 30, 40, 50, 60, 80, 100, 150, or 200 DNA base pairs apart). For purposes of gene editing, a CRISPR array can be designed to contain one or more guide RNA sequences corresponding to a desired target DNA sequence; see, e.g., cong et al (2013) Science [ Science ],339:819-823; ran et al (2013) Nature Protocols [ Nature laboratory Manual ],8:2281-2308. Cas9 requires at least 16 or 17 nucleotides of the gRNA sequence for DNA cleavage to occur; for Cpf1, at least 16 nucleotides of the gRNA sequence are required to achieve detectable DNA cleavage, and it is reported that for efficient DNA cleavage in vitro, at least 18 nucleotides of the gRNA sequence are required; see Zetsche et al (2015) Cell [ Cell ],163:759-771. In practice, guide RNA sequences are typically designed to have a length of 17-24 nucleotides (typically 19, 20 or 21 nucleotides) and to be precisely complementary (i.e., fully base-paired) to the targeted gene or nucleic acid sequence; guide RNAs that have less than 100% complementarity to the target sequence (e.g., grnas that are 20 nucleotides in length and have 1-4 mismatches to the target sequence) may be used, but may increase the likelihood of off-target effects. The design of effective guide RNAs for plant genome editing is disclosed in U.S. patent application publication 2015/0082478A1 (the entire specification of which is incorporated herein by reference). Recently, effective gene editing has been achieved using chimeric "single guide RNAs" ("sgrnas") (an engineered (synthetic) single RNA molecule that mimics a naturally occurring crRNA-tracrRNA complex and contains tracrRNA (for binding nucleases) and at least one crRNA (to guide nucleases to sequences targeted for editing); see, e.g., cong et al (2013) Science [ Science ],339:819-823; xing et al (2014) BMC Plant Biol [ BMC Plant Biol ],14:327-340. Chemically modified sgrnas have proven to be effective in genome editing; see, e.g., hendel et al (2015) Nature Biotechnol [ natural biotechnology ],985-991. The design of effective grnas for plant genome editing is disclosed in U.S. patent application publication 2015/0082478A1 (the entire specification of which is incorporated herein by reference).

Genomic DNA may also be modified by base editing. Adenine Base Editor (ABE) to convert A/T base pairs to G/C base pairs in genomic DNA and cytosine base pair editor (CBE) to effect C to T substitution can be used in certain embodiments of the methods provided herein. In certain embodiments, useful ABEs and CBEs can comprise genomic site-specific DNA binding elements (e.g., RNA-dependent DNA binding proteins, including catalytically inactivated Cas9 and Cas12 proteins or Cas9 and Cas12 nickases) operably linked to adenine or cytidine deaminase and used with guide RNAs that localize the protein near the nucleotide to be targeted for substitution. Suitable ABE and CBE's disclosed in the literature (Kim, nat Plants [ Natural Plants ], month 3 of 2018; 4 (3): 148-151) are suitable for use in the methods described herein. In certain embodiments, the CBE may comprise a fusion between a catalytically inactive Cas9 (dCas 9) RNA-dependent DNA binding protein fused to a cytidine deaminase that converts cytosine (C) to uridine (U) and a selected guide RNA, thereby achieving C-to-T substitution; see Komor et al (2016) Nature, 533:420-424. In other embodiments, the substitution of C to T is effected by Cas9 nickase [ Cas9n (D10A) ] fused to the modified cytidine deaminase and optionally phage μdsdna (double stranded DNA) end-binding protein Gam; see Komor et al, sci Adv [ science front ] month 8 of 2017; 3 (8) eaao4774. In other embodiments, an Adenine Base Editor (ABE) comprising adenine deaminase fused to a catalytically inactive Cas9 (dCAS 9) or Cas 9D 10A nickase may be used to convert A/T base pairs in genomic DNA to G/C base pairs (Gaudelli et al, (2017) Nature [ Nature ]551 (7681): 464-471).

In certain embodiments, zinc finger nucleases or zinc finger nickases may 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, each of which recognizes 9 to 18 base pairs, and a DNA cleavage domain comprising a nuclease domain (typically Fokl). The cleavage domain dimerizes to cleave DNA; thus, a pair of ZFNs is needed to target non-palindromic target polynucleotides. In certain embodiments, the described zinc finger nucleases and zinc finger nicking enzyme design methods (Urnov et al (2010) Nature Rev. Genet. [ Nature reviewed genet ],11:636-646; mohanta et al (2017) Genes [ Gene ]. 8, 12:399; ramirez et al Nucleic Acids Res. [ nucleic acids research ] (2012); 40 (12): 5560-5568; liu et al (2013) Nature Communications [ Nature communication ], 4:2565) may be adapted for use in the methods described herein. The zinc finger binding domain of a zinc finger nuclease or nicking enzyme provides specificity and can be engineered to specifically recognize any desired target DNA sequence. The zinc finger DNA binding domain is derived from a large class of DNA binding domains of eukaryotic transcription factors known as Zinc Finger Proteins (ZFPs). The DNA binding domain of ZFP typically contains a tandem array of at least three zinc "fingers," each recognizing a particular DNA triplet. A number of strategies can be used to design the binding specificity of zinc finger binding domains. One method, known as "modular assembly", relies on the functional autonomy of a single zinc finger with DNA. In this approach, a given sequence is targeted by identifying the zinc fingers of each triplet component in the sequence and ligating them into a multi-fingered 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 nucleotide bases beyond adjacent fingers and their target triplets. Typically, the engineered zinc finger DNA binding domain has a new binding specificity compared to naturally occurring zinc finger proteins. Engineering methods include, for example, rational design and various types of selection. Rational design includes, for example, the use of a database of triplex (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, wherein each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of a zinc finger that bind to a particular triplet or quadruplet sequence. See, for example, U.S. Pat. nos. 6,453,242 and 6,534,261, each of which is incorporated herein by reference in its 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, enhancing the binding specificity of zinc finger binding domains has been described in U.S. patent 6,794,136, which is incorporated herein by reference in its entirety. In addition, any suitable linker sequence may be used to join together the individual zinc finger domains. Examples of linker sequences are well known, see for example us patent 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 the DNA. Thus, cleavage by Fokl as part of ZFN requires two adjacent and independent binding events that must occur in the correct orientation and at the appropriate intervals to allow dimer formation. The requirement for two DNA binding events enables a more specific targeting of long and potentially unique recognition sites. Fokl variants with enhanced activity have been described and may be suitable for use in the methods described herein; see, e.g., guo et al (2010) J.mol.biol. [ journal of molecular biology ],400:96-107.

Transcription activator-like effectors (TALEs) are proteins secreted by certain Xanthomonas (Xanthomonas) species to regulate gene expression in host plants and promote bacterial colonization and survival. TALEs act as transcription factors and regulate expression of resistance genes in plants. Recent studies on TALEs reveal the codons linking the repeat region of TALEs to their target DNA binding sites. TALEs comprise highly conserved and repetitive regions consisting of tandem repeats of most 33 or 34 amino acid segments. Repeat monomers differ from each other mainly at amino acid positions 12 and 13. A strong correlation has been found between the unique pairs of amino acids at positions 12 and 13 and the corresponding nucleotides in the TALE binding site. The simple relationship between amino acid sequence and DNA recognition of TALE binding domains allows the design of DNA binding domains of any desired specificity. TALEs can be linked to non-specific DNA cleavage domains to produce genomic editing proteins, known as TAL effector nucleases or TALENs. As in the case of ZFNs, restriction endonucleases such as Fokl can be conveniently used. Methods of using 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 [ Proc. Natl. Acad. Sci. USA, 108:2623-2628; mahfouz (2011) GM crop [ transgenic crop ],2:99-103; mohanta et al (2017) Genes [ Gene ] volume 8, 12:399.TALE nicking enzymes have also been described and can be adapted for use in the methods described herein (Wu et al; biochem Biophys Res Commun. [ communication of biochemistry and biophysics research ] (2014); 446 (1): 261-6; luo et al; scientific Reports [ science report ]6, article number: 20657 (2016)).

Examples of donor DNA template molecules having sequences integrated at the site of at least one Double Strand Break (DSB) in the genome include double-stranded DNA, single-stranded DNA/RNA hybrids, and double-stranded DNA/RNA hybrids. In embodiments, the donor DNA template is provided directly to the plant protoplast or plant cell as a double-stranded (e.g., dsDNA or dsDNA/RNA hybrid) molecule in the form of double-stranded DNA or double-stranded DNA/RNA hybrid, or as two single-stranded DNA (ssDNA) molecules capable of hybridizing to form dsDNA, or as a single-stranded DNA molecule and a single-stranded RNA (ssRNA) molecule capable of hybridizing to form double-stranded DNA/RNA hybrid; that is, double stranded polynucleotide molecules are not provided indirectly, for example, by expression of dsDNA encoded by a plasmid or other vector in a cell. In various non-limiting embodiments of the method, the donor DNA template molecule that is integrated (or has an integrated sequence) at least one Double Strand Break (DSB) site in the 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" at one or both ends consisting of unpaired nucleotides (e.g., 1, 2, 3, 4, 5, or 6 unpaired nucleotides). In embodiments, the DSBs in the genome have no unpaired nucleotides at the cleavage site, and the donor DNA template molecule that is integrated at the DSB site (or has a sequence that is integrated at the DSB site) is a blunt-ended double-stranded DNA or a 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 DSBs in the genome have one or more unpaired nucleotides on one or both sides of the cleavage site, and the donor DNA template molecule that is integrated at the DSB site (or has a sequence that is integrated at the DSB site) is a double-stranded DNA or double-stranded DNA/RNA hybrid molecule having an overhang or "sticky end" consisting of unpaired nucleotides at one or both ends, or alternatively is a single-stranded DNA or single-stranded DNA/RNA hybrid molecule; in an embodiment, the donor DNA template molecule DSB is a double-stranded DNA or double-stranded DNA/RNA hybrid molecule comprising an overhang at one or both ends, wherein the overhang consists of the same number of unpaired nucleotides as the number of unpaired nucleotides generated at the site of the DSB by a nuclease that cleaves in an offset manner (wherein the Cas12 nuclease implements an offset DSB with a 5 nucleotide overhang in the genomic sequence, is to be integrated into the DSB site (or has a sequence to be integrated into the DSB site), and has 5 unpaired nucleotides at one or both ends). In certain embodiments, one or both ends of the donor DNA template molecule do not comprise regions of sequence homology (identity or complementarity) to genomic regions flanking the DSB; that is, one or both ends of the donor DNA template molecule do not contain sequence regions that are sufficiently complementary to allow hybridization with genomic regions immediately adjacent to the DSB location. In an embodiment, the donor DNA template molecule does not comprise homology to the DSB locus, that is, the donor DNA template molecule does not comprise a nucleotide sequence that is sufficiently complementary to allow hybridization to a genomic region immediately adjacent to the DSB locus. In embodiments, the donor DNA template molecule is at least partially double-stranded and comprises 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, consisting 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 comprises 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 has at least one overhang at one or both ends or a "sticky end" consisting of at least one additional unpaired nucleotide. In one embodiment, the donor DNA template molecule integrated at least one double-strand break (DSB) site in the genome (or having a sequence integrated at least one double-strand break (DSB) site in the genome) is a blunt-ended double-stranded DNA or a blunt-ended double-stranded DNA/RNA hybrid molecule having 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 linkage between adjacent nucleotides at the 5 'end, the 3' end, or both the 5 'and 3' ends. In embodiments, the donor DNA template molecule comprises 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 least 280, or at least 320 nucleotides. In embodiments, the donor DNA template molecule is 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 about 2 to about 320 base pairs (if double-stranded) or nucleotides (if single-stranded), or about 2 to about 500 base pairs (if double-stranded) or nucleotides (if single-stranded), or about 5 to about 300 base pairs (if double-stranded) or nucleotides (if single-stranded), or about 11 to about 300 base pairs (if double-stranded) or nucleotides (if single-stranded), or about 18 to about 300 base pairs (if double-stranded) or nucleotides (if single-stranded), or about 30 to about 100 base pairs (if double-stranded) or nucleotides (if single-stranded). In embodiments, the donor DNA template molecule comprises chemically modified nucleotides (see, e.g., various modifications of internucleotide linkages, bases, and sugars described in Verma and Eckstein (1998) annu. Rev. Biochem. [ biochemical yearbook ], 67:99-134); in embodiments, the naturally occurring phosphodiester backbone of the donor DNA template molecule is partially or fully modified with phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications, or the donor DNA template molecule comprises a modified nucleobase or modified sugar, or the donor DNA template molecule is labeled with a fluorescent moiety (e.g., fluorescein or rhodamine or a fluorescent nucleoside analog) or other detectable label (e.g., biotin or an isotope). In another embodiment, the donor DNA template molecule comprises a 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 DNA donor molecules (including single-stranded, chemically modified DNA donor molecules) that are integrated at (or have sequences integrated at) a double-stranded break site in a similar procedure.

The donor DNA template molecules used in the methods provided herein include DNA molecules comprising a first homology arm, a replacement DNA, and a second homology arm from 5 'to 3', wherein the homology arm contains sequences that are partially or completely homologous to genomic DNA (gDNA) sequences flanking a target site specific endonuclease cleavage site in gDNA. In certain embodiments, the replacement DNA may comprise 1 or more DNA base pair insertions, deletions or substitutions relative to the target gDNA. In embodiments, the donor DNA template molecule is double-stranded and is fully base-paired over all or most of its length, with the possible exception of any unpaired nucleotides at either or both ends. In another embodiment, the donor DNA template molecule is double-stranded and comprises one or more non-end mismatched or unpaired nucleotides in an additional double-stranded duplex. In embodiments, the donor DNA template molecule integrated at least one Double Strand Break (DSB) site comprises 2-20 nucleotides in one strand (if single stranded) or 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 both strands, each of which can base pair with a nucleotide on the opposite strand (in the case of a fully base paired double stranded polynucleotide molecule). Such donor DNA templates may be integrated into genomic DNA containing flat and/or staggered double-stranded DNA breaks by Homology Directed Repair (HDR). In certain embodiments, the donor DNA template homology arms may be about 20, 50, 100, 200, 400, or 600 to about 800 or 1000 base pairs in length. In certain embodiments, the donor DNA template molecule can be delivered to the plant cell in a circular (e.g., plasmid or viral vector, including geminivirus vector) or linear DNA molecule. In certain embodiments, the circular or linear DNA molecules used may comprise a modified donor DNA template molecule comprising, from 5 'to 3', a first copy of a target sequence specific endonuclease cleavage site sequence, a first homology arm, a replacement DNA, a second homology arm, and a second copy of a 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 eukaryotic cells to release an HDR-mediated genomic modified donor DNA template molecule that can participate in a target editing site in the plant cell genome. In certain embodiments, the donor DNA template may comprise a linear DNA molecule comprising, from 5 'to 3', a cleaved target sequence specific endonuclease cleavage site sequence, a first homology arm, a replacement DNA, a second homology arm, and a cleaved target sequence specific endonuclease cleavage site sequence. In certain embodiments, the cleaved target sequence-specific endonuclease sequence may comprise the following: blunt DNA ends or blunt DNA ends optionally comprising a 5' phosphate group. In certain embodiments, the cleaved target sequence-specific endonuclease sequence comprises a DNA terminus having a single-stranded 5 'or 3' DNA overhang. The target sequence specific endonuclease cleavage site sequence for such cleavage can be generated by: cleavage of the complete target sequence specific endonuclease cleavage site sequence or synthesis of a copy of the cleaved target sequence specific endonuclease cleavage site sequence. The donor DNA template may be chemically synthesized or enzymatically synthesized (e.g., in a Polymerase Chain Reaction (PCR)).

Various treatments may be used to deliver gene editing molecules and/or other molecules to plant cells. In certain embodiments, one or more treatments are employed to deliver gene edits or other molecules (e.g., comprising polynucleotides, polypeptides, or combinations thereof) into eukaryotic cells or plant cells, e.g., across a barrier such as a cell wall, plasma membrane, nuclear envelope, and/or other lipid bilayer. In certain embodiments, a polynucleotide, polypeptide, or RNP-containing composition comprising a molecule is delivered directly, for example, by contacting the composition directly with a plant cell. The foregoing compositions may be provided in the form of a liquid, solution, suspension, emulsion, inverse emulsion, colloid, dispersion, gel, liposome, micelle, injectable material, aerosol, solid, powder, microparticle, nanoparticle, or combination thereof, which may be applied directly to a plant, plant part, plant cell, or plant explant (e.g., by abrasion or puncturing or otherwise disrupting cell walls or cell membranes, by spraying or dipping or soaking or otherwise contacting directly, by microinjection). For example, a plant cell or plant protoplast is immersed in a composition comprising a liquid genome editing molecule, thereby delivering an agent to the plant cell. In certain embodiments, the agent-containing composition is delivered using negative or positive pressure, for example, using vacuum infiltration or applying hydrodynamic or fluid pressure. In certain embodiments, the agent-containing composition is introduced into the plant cell or plant protoplast, e.g., by microinjection or by rupture or deformation of the cell wall or cell membrane, e.g., by physical treatment, such as by application of negative or positive pressure, shear forces, or with a chemical or physical delivery agent such as a surfactant, liposome, or nanoparticle; see, for example, U.S. published patent application 2014/0287509, incorporated herein by reference in its entirety, for delivery of materials to cells through cell deformation constriction using microfluidic flow. Other techniques that may be used to deliver the agent-containing composition to eukaryotic cells, plant cells, or plant protoplasts include: ultrasonic or ultrasonic treatment; vibration, friction, shear stress, vortex, cavitation; centrifuging or applying mechanical force; mechanical cell wall or membrane deformation or rupture; enzymatic cell wall or cell membrane rupture or permeabilization; abrasion or mechanical scoring (e.g., abrasion with silicon carbide or other particulate abrasive or scoring with file or sandpaper) or chemical scoring (e.g., treatment with acid or caustic); electroporation. In certain embodiments, the agent-containing composition is provided by transfecting a plant cell or plant protoplast with a polynucleotide bacterium that encodes a genome editing molecule (e.g., an RNA-dependent DNA endonuclease, an RNA-dependent DNA binding protein, an RNA-dependent nicking enzyme, ABE or CBE, and/or a guide RNA) (e.g., agrobacterium (Agrobacterium sp.), rhizobium (Rhizobium sp.), sinorhizobium (Sinorhizobium sp.), mesorhizobium (Mesorhizobium sp.), bradyrhizobium (Bradyrhizobium sp.), azotobacter sp.), phyllobacterium (Phyllobacterium sp.); see, e.g., broothaerts et al (2005) Nature, 433:629-633). Any one of these techniques or a combination thereof may alternatively be used with plant explants, plant parts or tissues or whole plants (or seeds), from which plant cells are then optionally obtained or isolated; in certain embodiments, the agent-containing composition is delivered in a separate step after isolation of the plant cells.

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 cells in a plant. In certain embodiments, the polynucleotide vector comprises a regulatory element, such as a promoter operably linked to one or more polynucleotides encoding a genome editing molecule and/or a trait conferring gene. In such embodiments, expression of these polynucleotides can be controlled by selection of an appropriate promoter, particularly one that is functional in eukaryotic cells (e.g., plant cells); useful promoters include constitutive, conditional, inducible and time or space specific promoters (e.g., tissue specific promoters, developmentally regulated promoters or cell cycle regulated promoters). Developmental regulatory promoters useful in plant cells include phosphophosphotransferase protein (PLTP), fructose-1, 6-bisphosphatase protein, NAD (P) -bound Rossmann Fold (Rossmann-Fold) protein, adipocyte plasma membrane-associated protein-like protein, rieske [2Fe-2S ] iron-sulfur domain protein, chloroplast respiration (chloropirate) reduction 6 protein, D-glycerate 3-kinase, chloroplast-like protein, chlorophyll a-B binding protein 7, chloroplast-like protein, ultraviolet B-inhibitory protein, soul (Soul) heme binding family protein, photosystem I reactive central subunit psi-N protein, and dehydrogenase/reductase protein, as disclosed in U.S. patent application publication No. 20170121722 (incorporated herein by reference in its entirety and specifically for such disclosure). In certain embodiments, the promoter is operably linked to a nucleotide sequence encoding a plurality of guide RNAs, wherein the sequences encoding the guide RNAs are separated by cleavage sites (e.g., nucleotide sequences encoding microrna recognition/cleavage sites or self-cleaving ribozymes) (see, e.g., ferre-D' amare and Scott (2014) Cold Spring Harbor Perspectives Biol. [ cold spring harbor view in biology ],2: a 003574). In certain embodiments, the promoter is an RNA polymerase III promoter operably linked to a nucleotide sequence encoding one or more guide RNAs. In certain embodiments, the RNA polymerase III promoter is a plant U6 spliceosome RNA promoter, which may be native to the plant cell genome or from a different species, e.g., a U6 promoter from maize, tomato, or soybean, such as the promoters disclosed in U.S. patent application publication 2017/0166912, or homologs thereof; in an example, such a promoter is operably linked to a DNA sequence encoding a first RNA molecule (including Cas12a gRNA), followed by an operably linked 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 a chimera thereof, e.g., as described in U.S. patent application publication 20170166912. In certain embodiments, the promoter operably linked to the one or more polynucleotides is a constitutive promoter driving expression of a gene in a eukaryotic cell (e.g., a plant cell). In certain embodiments, the promoter drives gene expression in the nucleus or an organelle (e.g., chloroplast or mitochondria). Examples of constitutive promoters for plants include the CaMV 35S promoter disclosed in U.S. Pat. Nos. 5,858,742 and 5,322,938, the rice actin promoter disclosed in U.S. Pat. No. 5,641,876, the maize chloroplast aldolase promoter disclosed in U.S. Pat. No. 7,151,204, and 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 the genome editing system is a promoter from Fig Mosaic Virus (FMV), a RUBISCO promoter, or a Pyruvate Phosphate Dikinase (PPDK) promoter, which is active in photosynthetic tissue. Other contemplated promoters include cell-specific or tissue-specific or developmentally regulated promoters, e.g., promoters that limit expression of the nucleic acid targeting system to a germ line cell or germ line cell (e.g., promoters encoding DNA ligases, recombinases, replicases, or other genes specifically expressed in a germ line cell or germ line cell). In certain embodiments, genomic alterations are limited to only those cells from which DNA is inherited in the offspring, which is advantageous where it is desirable to limit expression of the genome editing system to avoid genotoxicity or other unwanted effects. All patent publications cited in this paragraph are incorporated herein by reference in their entirety.

The expression vectors or polynucleotides provided herein may comprise a DNA segment near the 3' end of the expression cassette that serves as a signal to terminate transcription and direct polyadenylation of the resulting mRNA, and may also support promoter activity. Such 3' elements are commonly referred to as "3' -untranslated regions" or "3' -UTRs" or "terminators" or "polyadenylation signals". In some cases, the 3 'element (or terminator) based on a plant gene consists of a 3' -UTR and a downstream non-transcribed sequence (Nuccio et al, 2015). Useful 3' elements include: the Agrobacterium tumefaciens nos 3', tml 3', tmr 3', tms 3', ocs 3' and tr7 3' elements disclosed in U.S. patent No. 6,090,627 (incorporated herein by reference), and the 3' elements from plant genes (e.g., heat shock protein 17, ubiquitin and fructose-1, 6-bisphosphatase genes from wheat (Triticum aestivum), and gluten, lactate dehydrogenase and beta-tubulin genes from rice (Oryza sativa)) disclosed in U.S. patent application publication 2002/0192813A 1. All patent publications cited in this paragraph are incorporated herein by reference in their entirety.

In certain embodiments, the plant cells may comprise haploid, diploid or polyploid plant cells or plant protoplasts, such as those obtained from haploid, diploid or polyploid plants, plant parts or tissues or calli. In certain embodiments, the plant cells in culture (or regenerated plants, progeny seeds, and progeny plants) 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, for example, methods for producing haploids in arabidopsis by crossing wild type strains with haploid inducer strains expressing altered forms of centromere-specific histone CENH3, as described below: maruthachalam and Chan in "How to make Haploid Arabidopsis thaliana [ how to make Haploid Arabidopsis ]," available at www [ dot ] openwet [ dot ] org/images/d/d3/haploid_Arabidopsis_protocol [ dot ] pdf; (Ravi et al (2014) Nature Communications [ Nature communication ],5:5334, doi:10.1038/ncomms 6334). Haploids can also be obtained in a wide variety of monocots (e.g., maize, wheat, rice, sorghum, barley) or dicots (e.g., soybean, brassica species (Brassica sp.), including canola, cotton, tomato) by crossing plants comprising a mutated CENH3 gene with wild-type diploid plants to produce haploid offspring, as disclosed in U.S. patent No. 9,215,849, which is incorporated herein by reference in its entirety. Haploid induced maize lines that can be used to obtain haploid maize plants and/or cells include Stock 6, MHI (morkow haploid inducer (Moldovian Haploid Inducer)), indeterminate gametophyte (ig) mutations, KEMS, RWK, ZEM, ZMS, KMS, and transgenic haploid inducer lines disclosed in U.S. 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 tissue, e.g., from flowers, developing flowers or flower buds, ovaries, ovules, megaspores, anthers, pollen, megagametophytes, 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., spontaneous chromosome doubling by meiosis is not reduced, or by using chromosome doubling agents such as colchicine, acesulfame, trifluralin, naphal, nitrous oxide gas, antimicrotubule herbicides, antimicrotubule agents, and mitosis inhibitors) in the plant cell or plant protoplast to produce a doubled haploid plant cell or plant protoplast in which the complement of the gene or allele is homozygous; still other embodiments include regenerating a doubled haploid plant from a doubled haploid plant cell or plant protoplast. Another embodiment relates to a hybrid plant having at least one parent plant that is a doubled haploid plant provided by the method. The generation of doubled haploid plants provides homozygosity within a generation without the need for several generations of selfing to obtain homozygous plants. The use of doubled haploids is advantageous in any situation where it is desirable to establish genetic purity (i.e., homozygosity) in as short a time as possible. Doubled haploid production is particularly advantageous in slow growing plants, or is particularly useful for producing hybrid plants that are offspring of at least one doubled haploid plant.

In certain embodiments, the plant cells used in the methods provided herein can include non-dividing cells. Such non-dividing cells may include plant cell protoplasts, plant cells that have been subjected to one or more genetic and/or drug-induced cell cycle blockages, and the like.

In certain embodiments, plant cells for use in the methods provided herein can comprise dividing cells. Dividing cells may include those found in various plant tissues including leaves, meristems, and embryos. These tissues include, but are not limited to, dividing cells from maize young leaves, meristematic tissue, and scutellum tissue (from embryos from about 8 or 10 to about 12 or 14 Days After Pollination (DAP)). The isolation of maize embryos has been described in several publications (Brettschneider, becker, and

1997; leduc et al 1996; frame et al 2011; wang and Frame 2009). In certain embodiments, basal leaf tissue (e.g., leaf tissue located about 0 to 3cm from the leaf tongue of maize plants; kirienko, luo, and Sylvester 2012) is a target for HDR-mediated gene editing. Methods for obtaining regenerable plant structures and regenerated plants from HDR-mediated plant cell gene editing provided herein can be used To adapt the method disclosed in U.S. patent application publication No. 20170121722, which is incorporated by reference in its entirety and specifically with respect to such disclosure. In certain embodiments, a single plant cell subjected to HDR-mediated gene editing will produce a single regenerable plant structure. In certain embodiments, a single regenerable plant cell structure may be formed from a single cell on or within an explant that has undergone HDR-mediated gene editing.

In some embodiments, the methods provided herein may include the additional step of growing or regenerating a plant from a plant cell that has undergone improved HDR-mediated gene editing or from a regenerable plant structure obtained from the plant cell. In certain embodiments, the plants may further comprise an inserted transgene, target gene editing, or genome editing provided by the methods and compositions disclosed herein. In certain embodiments, the calli are produced by plant cells, and plantlets and plants are produced from such calli. In other embodiments, the entire seedling or plant grows directly from plant cells without the callus stage. Thus, further related aspects relate to whole seedlings and plants grown or regenerated from plant cells or plant protoplasts having target gene editing or genome editing, and seeds of such plants. In certain embodiments in which plant cells or plant protoplasts are subjected to genetic modification (e.g., genome editing by, for example, rdDe), the growing or regenerating plant exhibits a phenotype associated with the genetic modification. In certain embodiments, the growing or regenerating plant includes two or more genetic or epigenetic modifications in its genome that in combination provide at least one phenotype of interest. In certain embodiments, a heterogeneous population of plant cells (at least some of which include at least one genetic or epigenetic modification) having a target gene editing or genome editing is provided by the method; related aspects include plants having a phenotype of interest associated with a genetic modification or an epigenetic modification, provided by: regenerating a plant having a phenotype of interest from a plant cell or plant protoplast selected from a heterogeneous population of plant cells having a target gene or genome editing, or selecting a plant having a phenotype of interest from a heterogeneous population of plant cells grown or regenerated from a population of plant cells having a target gene editing or genome editing. Examples of phenotypes of interest include herbicide resistance, improved tolerance to abiotic stress (e.g., tolerance to extreme temperatures, drought or salt) or biotic stress (e.g., resistance to nematodes, bacterial or fungal pathogens), improved nutrient or water utilization, modified lipid, carbohydrate or protein composition, improved flavor or appearance, improved storage characteristics (e.g., resistance to bruising (bruising), browning or softening), increased yield, altered morphology (e.g., floral structure or color, plant height, branching, root structure). In one embodiment, a heterogeneous population of plant cells (or seedlings or plants grown or regenerated therefrom) having target gene editing or genome editing is exposed to conditions that allow expression of a phenotype of interest; for example, selection for herbicide resistance may include exposing a population of plant cells (or seedlings or plants grown or regenerated therefrom) having a target gene editing or genome editing to an amount of herbicide or other growth-inhibiting or toxic substance, allowing identification and selection of those resistant plant cells (or seedlings or plants) that survive the treatment. Methods for obtaining and regenerating plants from plant cells or plant structures can be adapted from published procedures (Rosest and Gilissen, acta Bot. Neerl. [ Dutch plant theory ],1989,38 (1), 1-23; bhaskaran and Smith, crop Sci. [ Crop science ]30 (6): 1328-1337; ikeuchi et al, development ],2016, 143:1442-1451). The methods for obtaining and regenerating plants from plant cells or plant structures may also be adapted from U.S. patent application publication No. 20170121722 (incorporated herein by reference in its entirety and in particular with respect to such disclosure). Also provided are heterogeneous or homogeneous populations, subsequent generations, or seeds of such plants grown or regenerated from plant cells or plant protoplasts of such plants or parts thereof (e.g., seeds) having targeted gene editing or genome editing. Other related aspects include hybrid plants provided by crossing a first plant grown or regenerated from a plant cell or plant protoplast having target gene editing or genome editing and having at least one genetic or epigenetic modification with a second plant, wherein the hybrid plant comprises the genetic or epigenetic modification; seeds produced by hybrid plants are also contemplated. Also contemplated are progeny seeds and progeny plants, including hybrid seeds and hybrid plants having the regenerated plant as a parent or ancestor. The plant cells and derived plants and seeds disclosed herein can be used for a variety of purposes useful to the consumer or grower. In other embodiments, the processed product is made from a plant or seed thereof, comprising: (a) Corn, soybean, cotton or canola seed meal (defatted or not defatted); (b) extracted proteins, oils, sugars, and starches; (c) fermenting the product; (d) Animal feed or human food (e.g., feeds and foods comprising corn, soybean, cotton, or canola seed meal (defatted or not) and other ingredients (e.g., other grains, other seed meal, other protein meal, other oils, other starches, other sugars, binders, preservatives, humectants, vitamins, and/or minerals), (e) pharmaceuticals, (f) raw or processed biomass (e.g., cellulosic and/or lignocellulosic materials), and (g) various industrial products.

Examples

Various embodiments of plants, genomes, methods, biological samples and other compositions described herein are set forth in the following numbered sets of embodiments.

List of examples 1

1. An edited transgenic plant genome comprising a modification of an original transgenic locus, wherein the modification comprises a deletion of a segment of the original transgenic locus, the segment comprising, consisting essentially of, or consisting of: a synthetic cloning site sequence; replication of the transgene sequence; fragments of the transgene sequences; agrobacterium right and/or left border sequences;

and/or a segment of DNA that is not essential for the primary function of the transgenic locus and wherein optionally the primary function is herbicide tolerance, insect resistance, biofuel use, or male sterility.

2. The edited transgenic plant genome of example 1, wherein the modification of the original transgenic locus further comprises a deletion of a selectable marker gene, and optionally wherein the primary function is insect resistance, biofuel use, or male sterility.

3. An edited transgenic plant genome as described in example 1 or 2, wherein the modification comprises: (a) A deletion comprising, consisting essentially of, or consisting of the following segments: synthetic cloning sites, replication of the transgene sequence, fragments of the transgene sequence and/or agrobacterium right and/or left border sequences; or (b) a deletion of a selectable marker gene.

4. The edited transgenic plant genome of example 3, wherein the selectable marker gene confers antibiotic resistance, herbicide tolerance, or the ability to grow on a specific carbon source, wherein the specific carbon source is optionally mannose and optionally wherein the primary function is insect resistance, biofuel use, or male sterility.

5. The edited transgenic plant genome of example 4, wherein the selectable marker gene comprises DNA encoding: phosphinothricin Acetyl Transferase (PAT), glyphosate tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and Glyphosate Oxidase (GOX), neomycin phosphotransferase (npt), hygromycin phosphotransferase, aminoglycoside adenyltransferase or phosphomannose isomerase (pmi).

6. The edited transgenic plant genome of any of embodiments 1-5, wherein the length of the deleted segment of the original transgenic locus is at least two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 base pair DNA.

7. The edited transgenic plant genome of any one of claims 1-5, wherein the length of the deleted segment of the original transgenic locus is any of two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, 40, 45, or 50 to 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, or 900 base pair DNA and at least two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, 40, 45, or 50 to 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1,000 base pair DNA; optionally, the original transgene locus in which it is deleted is 10 to 500 DNA base pairs in length.

8. The edited transgenic plant genome of any of embodiments 1-7 wherein the deletion segment is flanked by operably linked Protospacer Adjacent Motif (PAM) sites in the original transgenic locus and/or wherein the deletion segment comprises operably linked PAM sites in the original transgenic locus.

9. The edited transgenic plant genome of any one of embodiments 1-8 comprising PAM sites flanking the deletion site in the modified transgenic locus.

10. The edited transgenic plant genome of any one of embodiments 1-9, wherein the PAM site is recognized by an RNA-dependent DNA endonuclease (RdDe); optionally wherein the RdDe is a type II or type V RdDe type 2.

11. The edited transgenic plant genome of any of embodiments 1-10, wherein the modification comprises two or more separate deletions.

12. The edited transgenic plant genome of any one of embodiments 1-11 wherein there are modifications in two or more original transgenic plant loci.

13. The edited transgenic plant genome of any of embodiments 1-12, wherein a deleted segment of the original transgenic locus is replaced in the modified transgenic locus by an introduced DNA sequence.

14. The edited transgenic plant genome of any of embodiments 1-13, wherein the modification comprises Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017, MON89034, MIR162, MIR604, NK603, SYN-E3272 in a transgenic maize plant genome-5、5307、DAS-40278、DP-32138、DP-33121、HCEM485、LY038、MON863、MON87403、MON87403、MON87419、MON87460、MZHG0JG、MZIR098、

98140 or TC1507 modification of the original transgene locus.

15. The edited transgenic plant genome of any of embodiments 1-13, wherein the modification comprises a5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788,

Or modification of the original transgene locus of SYHT0H 2.

16. The edited transgenic plant genome of any of embodiments 1-13, wherein the modification comprises modification of the original transgenic locus of DAS-21023-5, DAS-24236-5, COT102, LL cotton 25, MON15985, MON88701, or MON88913 in the transgenic cotton plant genome.

17. The edited transgenic plant genome of any one of embodiments 1-13, wherein the modification comprises modification of GT73, HCN28, MON88302, or MS8 original transgene locus in the transgenic canola plant genome.

18. A transgenic plant cell comprising the edited transgenic plant genome of any one of embodiments 1-17.

19. A transgenic plant comprising the edited transgenic plant genome of any one of embodiments 1-17.

20. A transgenic plant part comprising the edited transgenic plant genome of any one of embodiments 1-17.

21. The transgenic plant part of embodiment 20, wherein the plant part is a seed, leaf, tuber, stem, root, or pod.

22. A processed transgenic plant product obtained from the transgenic plant part of example 20 or 21, wherein the processed plant product comprises a polynucleotide comprising a portion of the modified transgenic locus comprising a deletion site of a segment of the original transgenic locus.

23. A biological sample obtained from a transgenic plant cell as described in example 18, a transgenic plant as described in example 19 or a transgenic plant part as described in example 20 or 21, wherein the biological sample comprises a polynucleotide comprising a portion of the modified transgene locus comprising a deletion site of a segment of the original transgene locus.

24. A method of editing a transgenic plant genome to obtain a plant cell, plant part or plant containing a modification of an original transgenic locus, the method comprising: (a) Contacting a transgenic plant genome with one or more gene-editing molecules that introduce one or more single-or double-strand breaks to provide excision of a segment of the original transgenic locus, the segment comprising, consisting essentially of, or consisting of: a synthetic cloning site sequence; replication of the transgene sequence; fragments of the transgene sequences; agrobacterium right and/or left border sequences; and/or a segment of DNA that is not essential for the primary function of the transgenic locus and wherein optionally the primary function is herbicide tolerance, insect resistance, biofuel use or male sterility, and (b) selecting a plant cell, plant part or plant containing the modified transgenic locus, wherein the segment comprising or consisting of: synthetic cloning site sequences, replication of transgene sequences, fragments of transgene sequences, agrobacterium right and/or left border sequences, and/or DNA segments not necessary for expression of any transgene in the locus, thereby obtaining a plant cell, plant part or plant containing the modified transgene locus; optionally wherein the transgenic plant genome is contacted in step (a) by introducing one or more compositions comprising or encoding these gene editing molecules into a plant cell comprising the transgenic plant genome.

25. The method of embodiment 24, wherein the method further comprises excision of DNA comprising the selectable marker gene from the original transgene locus.

26. The method of embodiment 24 or 25, wherein the segment comprising the synthetic cloning site sequence, the replication of the transgene sequence, the fragment of the transgene sequence and/or the agrobacterium right/left border sequence further comprises a selectable marker gene.

27. The method of any one of embodiments 24-26, further comprising contacting the genome with one or more gene-editing molecules that introduce one or more single-or double-strand breaks to provide excision of a selectable marker gene, wherein a segment comprising, consisting essentially of, or consisting of: the synthetic cloning site sequence, the replication of the transgene sequence, a fragment of the transgene sequence and/or the agrobacterium right and/or left border sequence; optionally selecting in step (b) a plant cell, plant part or plant containing the modified transgene locus, wherein the selectable marker gene and the segments comprising or consisting of: the synthetic cloning site sequence, replication of the transgene sequence, fragments of the transgene sequence, agrobacterium right and/or left border sequences, and/or segments of DNA not necessary for expression of any transgene in the locus.

28. The method of embodiment 26 or 27, wherein the selectable marker gene confers antibiotic resistance, herbicide tolerance, or the ability to grow on a specific carbon source, wherein the specific carbon source is optionally mannose.

29. The method of embodiment 28, wherein the selectable marker gene comprises DNA encoding: phosphinothricin Acetyl Transferase (PAT), glyphosate tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and Glyphosate Oxidase (GOX), neomycin phosphotransferase (npt), hygromycin phosphotransferase, aminoglycoside adenyltransferase or phosphomannose isomerase (pmi).

30. The method of any one of embodiments 24-29, wherein the transgenic plant cell is in a tissue culture, a callus culture, a plant part or whole plant.

31. The method of any one of embodiments 24-30, wherein the transgenic plant cell is a haploid plant cell, optionally wherein the plant is a haploid plant.

32. The method of any one of embodiments 24-31, wherein the length of the deleted segment of the original transgenic locus is at least two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 base pair DNA.

33. The method of any one of embodiments 24-31, wherein the length of the deleted segment of the original transgene locus is any of two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, 40, 45, or 50 to 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, or 900 base pair DNA and at least two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, 40, 45, or 50 to 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1,000 base pair DNA; optionally, wherein the deletion segment of the original transgene locus is between 10 and 500 base pairs in length of DNA.

34. The method of any one of embodiments 24 to 33, wherein the one or more gene editing molecules are selected from the group consisting of: RNA-dependent DNA endonucleases (RdDe) and/or guide RNAs, RNA-dependent nicking enzymes and/or guide RNAs, zinc finger nucleases or nicking enzymes, and TALE nucleases or nicking enzymes.

35. The method of any one of embodiments 24 to 34, wherein the deletion segment is flanked by operably linked Protospacer Adjacent Motif (PAM) sites in the original transgene locus and/or wherein the deletion segment comprises operably linked PAM sites in the original transgene locus.

36. The method of any one of embodiments 24-35, wherein the modified transgene locus comprises a PAM site flanking the deletion site.

37. The method of any one of embodiments 24 to 36, wherein the PAM site is recognized by an RNA-dependent DNA endonuclease (RdDe); optionally wherein the RdDe is a type II or type V RdDe type 2.

38. The method of any one of embodiments 24 to 37, wherein the modification comprises two or more deletions.

39. The method of any one of embodiments 24 to 38, wherein two or more original transgenic plant loci are modified.

40. The method of any one of embodiments 24-39, wherein the deleted segment of the original transgene locus is replaced in the modified transgene locus by an introduced DNA sequence.

41. The method of embodiment 40, wherein the gene editing molecules comprise a donor DNA template comprising the introduced DNA sequence,

optionally, wherein the transgenic plant cell, transgenic plant part or transgenic plant is selected for: the introduced DNA sequence is integrated into the original transgene locus at the deletion site of the deletion segment.

42. The method of any one of embodiments 24-41, wherein the modification comprises Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017, MON89034, MIR162, MIR604, NK603, SYN-E3272-5, 5307, DAS-40278, DP-32138, DP-33121, HCEM485, LY038, MON863, MON87403, MON87419, MON87460, mhg 0JG, mzi 098, in the genome of the transgenic maize plant,

98140 or TC1507 modification of the original transgene locus.

43. The method of any one of embodiments 24-41, wherein the modification comprises A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788,

or modification of the original transgene locus of SYHT0H 2.

44. The method of any one of embodiments 24-41, wherein the modification comprises modification of the original transgenic locus of DAS-21023-5, DAS-24236-5, COT102, LL cotton 25, MON15985, MON88701, or MON88913 in the genome of the transgenic cotton plant.

45. The method of any one of embodiments 24 to 41, wherein the modification comprises modification of GT73, HCN28, MON88302, or MS8 original transgene locus in the genome of a transgenic canola plant.

46. A method of obtaining a plant breeding line, the method comprising: (a) Crossing a transgenic plant comprising the edited transgenic plant genome of any one of examples 1-17 with a second plant; and (b) selecting from the cross a progeny plant comprising the modified transgene locus of the edited transgene genome, thereby obtaining a plant breeding line.

47. The method of embodiment 46, wherein the second plant further comprises an edited transgenic plant genome as in any one of embodiments 1-17; and (b) selecting from the cross a progeny plant comprising the modified transgene locus of the first plant and the modified transgene locus of the second plant, thereby obtaining a plant breeding line.

48. The method of embodiment 46 or 47, wherein the first plant or second plant further comprises an additional third modified transgene locus; and (b) selecting from the cross a progeny plant comprising the modified transgene locus of the first plant, the modified transgene locus of the second plant, and the third modified transgene locus, thereby obtaining a plant breeding line.

Examples

The following examples are provided for illustrative purposes only and are not intended to be limiting.

EXAMPLE 1 excision of selectable marker Gene from transgenic loci

The genome of a transgenic plant containing one or more of the following transgenic loci (events) with selectable marker genes is contacted with a type II or type 2V RdDe and a guide RNA that recognizes a designated target DNA site (guide RNA encoding plus PAM site) in the DNA segment flanking the selectable marker gene. Plant cells, calli, parts or whole plants are selected comprising a selectable marker gene deleted from a transgene locus in the genome of the transgenic plant.

TABLE 5 genomic DNA targets and PAM sites pre-existing in DNA flanking selectable marker genes for different events (transgene loci)

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EXAMPLE 2 excision of Agrobacterium left and Right border sequences from transgenic loci

The genome of a transgenic plant containing one or more of the following transgenic loci (events) with agrobacterium left and right border sequences is contacted with a class 2V RdDe and a guide RNA that recognizes a designated target DNA site (guide RNA coding plus PAM site) in the DNA segment flanking the agrobacterium right or side sequence. Plant cells, calli, parts or whole plants are selected comprising a selectable marker gene deleted from a transgene locus in the genome of the transgenic plant.

TABLE 6 genomic DNA targets and PAM sites pre-existing for class 2V Cas nucleases in DNA flanking the Agrobacterium right border sequence for different events (transgene loci)

TABLE 7 genomic DNA targets and PAM sites pre-existing for class 2V Cas nucleases in DNA flanking the Agrobacterium left border sequence for different events (transgene loci)

The genome of a transgenic plant containing one or more of the following transgenic loci (events) with agrobacterium left and right border sequences is contacted with a type II RdDe and a guide RNA that recognizes a designated target DNA site (guide RNA coding plus PAM site) in the DNA segment flanking the agrobacterium right or side sequence. Plant cells, calli, parts or whole plants are selected comprising a selectable marker gene deleted from a transgene locus in the genome of the transgenic plant.

TABLE 8 genomic DNA targets and PAM sites pre-existing for class 2 type II Cas nucleases in DNA flanking the Agrobacterium right border sequence for different events (transgene loci)

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TABLE 9 genomic DNA targets and PAM sites pre-existing for class 2 type II Cas nucleases in DNA flanking the Agrobacterium left border sequence for different events (transgene loci)

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The breadth and scope of the present disclosure should not be limited by any of the above-described embodiments.

Claims

1. A method of producing a elite crop plant comprising at least one modification of an approved transgene locus and at least one targeted genetic alteration conferring a desired trait, comprising the steps of:

(a) Transforming plant cells or plant tissue of a elite crop plant with a marker-based transformation system comprising at least one first selectable marker gene and one or more transgenes encoding at least one genome editing molecule, wherein the elite crop plant comprises a modification of the approved transgene locus, the modification comprising a deletion of a segment consisting essentially of or consisting of a second selectable marker gene, and wherein the genome editing molecule is designed to induce the at least one targeted genetic alteration;

(b) Selecting a plant comprising stable integration of the transformation system in the genomic DNA of the plant by using the first selectable marker gene, optionally wherein the first and second selectable marker genes confer the same selectable trait; and is also provided with

(c) Selecting a plant comprising the modification of the approved transgene locus and at least one targeted genetic alteration that confers the desired trait.

2. The method of claim 1, wherein the genome editing molecule comprises an RNA-dependent DNA endonuclease (RdDe) encoded by the transgene, one or more guide RNAs or polynucleotides encoding the guide RNAs, and optionally a donor DNA template.

3. The method of claim 2, wherein the transgene encoding the RNA-dependent DNA endonuclease (RdDe) is stably integrated into the genomic DNA, and wherein one or more guide RNAs or polynucleotides encoding the guide RNAs, and optionally a donor DNA template, are unstably integrated into the genomic DNA.

4. The method of claim 1, wherein transformation is achieved with agrobacterium, rhizobium, gene gun, nanotube or liposome transformation systems.

5. The method of claim 1, further comprising the step of selecting superior crop plants comprising modification of the approved transgene locus and the targeted genetic alteration, wherein the marker-based transformation system is absent.

6. The method of claim 1, comprising: (i) Selecting a plant in step (c) or in a progeny of a plant from step (c), wherein stable integration of the transformation system is absent.

7. A method of producing a elite crop plant comprising at least one modification of a transgene locus conferring a targeted genetic alteration and approval of a desired trait, comprising the steps of:

(i) Inducing at least one targeted genetic change in the maize plant genome with one or more genome editing molecules in a modified elite crop plant comprising the approved transgenic locus; and is also provided with

(ii) Selecting a modified elite crop plant comprising the approved transgene locus, wherein the targeted genetic alteration is present.

8. The method of claim 7, wherein the modification of the approved transgene locus comprises a deletion of a segment comprising, consisting essentially of, or consisting of a selectable marker gene.

9. The method of any one of claims 1 to 8, wherein the selectable marker gene comprises DNA encoding; (i) A gene that confers tolerance to a herbicide, optionally glyphosate or phosphinothricin; (ii) A gene encoding a gene conferring resistance to an antibiotic, optionally neomycin or hygromycin; or (iii) a gene capable of enabling the use of mannose as a carbon source; or (iv) Phosphinothricin Acetyl Transferase (PAT), glyphosate tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosate Oxidase (GOX), neomycin phosphotransferase (npt), hygromycin phosphotransferase (hpt), aminoglycoside adenyltransferase, or phosphomannose isomerase (pmi).

10. The method of claim 9, wherein the selectable marker gene comprises DNA encoding: phosphinothricin Acetyl Transferase (PAT), glyphosate Oxidase (GOX), neomycin phosphotransferase (npt), hygromycin phosphotransferase (hpt), aminoglycoside adenyltransferase or phosphomannose isomerase (pmi).

11. The method of any one of claims 1 to 8, wherein the modification of the approved transgene locus further comprises or comprises one or more deletions of a segment of the original approved transgene locus comprising, consisting essentially of, or consisting of:

synthetic cloning site elements;

transgenic;

replication of the transgenic element;

a fragment of the transgene;

agrobacterium right and/or left border elements;

and/or

Segments of DNA not necessary for expression of any transgene in the approved transgene locus;

optionally, wherein the replication and/or fragment of the transgene sequence is a promoter or a replication and/or fragment of a polyadenylation signal.

12. The method of any one of claims 1 to 8, wherein the modification of the approved transgene locus does not comprise a site-specific recombination system DNA recognition site, and optionally wherein the site-specific recombination system DNA recognition site is a lox or FRT site.

13. The method of any one of claims 1 to 8, wherein the modification of the approved transgene locus comprises a deletion of a segment consisting essentially of or consisting of a selectable marker gene, does not comprise a site-specific recombination system DNA recognition site, and optionally wherein the site-specific recombination system DNA recognition site is a lox or FRT site.

14. The method of any one of claims 1 to 8, wherein the deletion of the segment does not substantially affect other functional characteristics of the approved locus, and wherein the functional characteristics are optionally traits conferred by transgenes in the modified approved transgenic locus.

15. The method of any one of claims 1 to 8, wherein DNA segments 5 'and 3' of the original approved transgene locus comprising, consisting essentially of, or consisting of a deletion segment of a selectable marker gene are directly linked to form a selectable marker gene excision site in a modification in the original approved transgene locus.

16. The method of any one of claims 1-8, wherein the modification of the approved transgenic locus comprises Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017, MON89034, MIR162, MIR604, NK603, SYN-E3272-5, 5307, DAS-40278, DP-32138, DP-33121, HCEM485, LY038, MON863, MON87403, MON87419, MON87460, MZHG0JG, MZIR098,

Modification of the 98140 and/or TC1507 transgene locus.

17. The method of any one of claims 1-8, wherein the modification of the approved transgenic locus comprises a5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788,

and/or modification of the SYHT0H2 transgene locus.

18. The method of any one of claims 1 to 8, wherein the modification of the approved transgenic locus comprises modification of a DAS-21023-5, DAS-24236-5, COT102, LL cotton 25, MON15985, MON88701, and/or MON88913 transgenic locus in the genome of a transgenic cotton plant.

19. The method of any one of claims 1 to 8, the modification of the approved transgenic locus comprising modification of GT73, HCN28, MON88302 or MS8 transgenic locus in the genome of a transgenic canola plant.

20. The method of any one of claims 1 to 8, wherein the targeted genetic alteration confers a useful agronomic trait or quality trait.

21. The method of any one of claims 1 to 8, wherein the modification further comprises deleting, consisting essentially of, or consisting of a DNA segment comprising the nonessential DNA of the originally approved transgene locus.

22. A elite crop plant comprising a modification of an originally approved transgene locus, wherein the modification comprises a deletion of a segment comprising, consisting essentially of, or consisting of a selectable marker gene of the originally approved transgene locus, and wherein the modification of the approved transgene locus does not comprise a site specific recombination system DNA recognition site.

23. The elite crop plant according to claim 22, wherein the site-specific recombination system DNA recognition site is a lox or FRT site.

24. The elite crop plant according to claim 22, wherein the deletion of the segment does not substantially affect other functional characteristics of the approved locus, and wherein the functional characteristics are optionally traits conferred by transgenes in the modified approved transgene locus.

25. The elite crop plant according to claim 22, wherein DNA segments 5 'and 3' ofthe original approved transgene locus comprising, consisting essentially of, or consisting of a deletion segment are directly linked to form a selectable marker gene excision site in a modification in the original approved transgene locus.

26. The elite crop plant according to any one of claims 22 to 25, wherein the modification of the original approved transgene locus further comprises one or more deletions of a segment of the original approved transgene locus comprising, consisting essentially of, or consisting of:

synthetic cloning site elements;

transgenic;

replication of the transgenic element;

a fragment of the transgene;

agrobacterium right and/or left border elements;

and/or

27. The elite crop plant of any one of claims 22 to 25, wherein the elite crop plant further comprises at least one targeted genetic alteration conferring a desired trait.

28. The elite crop plant according to any one of claims 22 to 25, wherein the selectable marker gene comprises DNA encoding: phosphinothricin Acetyl Transferase (PAT), glyphosate tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosate Oxidase (GOX), neomycin phosphotransferase (npt), hygromycin phosphotransferase (hpt), aminoglycoside adenyltransferase or phosphomannose isomerase (pmi).

29. The elite crop plant according to claim 28, wherein the selectable marker gene comprises DNA encoding: phosphinothricin Acetyl Transferase (PAT), glyphosate Oxidase (GOX), neomycin phosphotransferase (npt), hygromycin phosphotransferase (hpt), aminoglycoside adenyltransferase or phosphomannose isomerase (pmi).

30. The elite crop plant of any one of claims 22 to 25, wherein the modification of the approved transgene locus comprises Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017, MIR162, MIR604, NK603, SYN-E3272-5, 5307, DAS-40278, DP-32138, DP-33121, HCEM485, MON863, MON87403, MON87419, MON87460, mhg 0JG, mzi 098, MON87,

Modification of the 98140 and/or TC1507 transgene locus.

31. The elite crop plant according to any one of claims 22 to 25, wherein the modification of the approved transgene locus comprises a5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788,

and/or modification of the SYHT0H2 transgene locus.

32. The elite crop plant according to any one of claims 22 to 25, wherein the modification of the approved transgenic locus comprises modification of a DAS-21023-5, DAS-24236-5, COT102, LL cotton 25, MON15985, MON88701, and/or MON88913 transgenic locus in the genome of the transgenic cotton plant.

33. The elite crop plant according to any one of claims 22 to 25, modification of the approved transgene locus comprises modification of the GT73, HCN28, MON88302 or MS8 transgene locus in the genome of a transgenic canola plant.

34. The elite crop plant of any one of claims 22 to 25, wherein the plant further comprises: (i) an additional original approved transgene locus; and/or (ii) an additional approved transgene locus comprising one or more modifications.

35. The elite crop plant according to any one of claims 22 to 25, wherein the modification of the original approved transgene locus further comprises a deletion of a DNA segment comprising, consisting essentially of, or consisting of the non-essential DNA of the original approved transgene locus.

36. An edited crop plant genome comprising a genome of a crop plant of any one of claims 22 to 25.

37. A method of obtaining the elite crop plant as claimed in any one of claims 22 to 25, the method comprising the steps of: (a) Obtaining a modified crop plant comprising an originally-approved transgene locus, the modification comprising a deletion of a selectable marker gene, a segment consisting essentially of, or a combination thereof comprising the originally-approved transgene locus, wherein the plant does not comprise the germplasm of the elite crop plant; and (b) introgressing the modified transgenic locus into the germplasm of the elite crop plant.

38. The method of claim 37, wherein the introgression comprises: (i) Crossing the crop plant of (a) with a plant comprising elite crop germplasm but lacking the modified transgene locus; (ii) Selecting a progeny plant comprising the modified transgenic locus; (iii) Backcrossing the progeny plant with a plant comprising the elite crop germplasm but lacking the modified transgene locus; and (iv) selecting a progeny plant comprising the modified transgenic locus.

39. A method of obtaining a population of large inbred seeds for commercial seed production, the method comprising selfing the elite crop plant according to any one of claims 22 to 25 and harvesting seed from the selfed elite crop plant.

40. A method of obtaining hybrid seed, the method comprising crossing a first plant comprising the edited genome of claim 36 with a second plant and harvesting seed from the crossing.

41. The method of claim 40, wherein the first plant and the second plant are in different sets of heterosis.

42. The method of claim 40 or 41, wherein the first or second plant is a pollen receptor that has become male sterile.

43. The method of claim 42, wherein the plant is rendered male sterile by emasculation, cytoplasmic male sterility, chemical crossing agents or systems, transgenes, and/or mutations in endogenous plant genes.

44. The method of claims 40-41, further comprising the step of sowing the hybrid seed.

45. A DNA comprising a selectable marker gene excision site wherein a selectable marker gene, consisting essentially of, or a segment consisting of the original approved transgene locus is deleted.

46. The DNA of claim 45, wherein the originally-approved transgene locus is Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017, MIR162, MIR604, NK603, SYN-E3272-5, 5307, DAS-40278, DP-33121, HCEM485, MON863, MON87403, MON87419, MON87460, MZIG 0JG, MZIR098,

98140 and/or TC1507 transgene locus.

47. The DNA according to claim 45, wherein the originally approved transgene locus is A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788,

And/or a SYHT0H2 transgene locus.

48. The DNA of claim 45, wherein the original approved transgene locus is a DAS-21023-5, DAS-24236-5, COT102, LL cotton 25, MON15985, MON88701, and/or MON88913 transgene locus.

49. The DNA of claim 45, wherein the originally approved transgene locus is the GT73, HCN28, MON88302, or MS8 transgene locus.

50. The DNA of any one of claims 45 to 49, wherein the DNA is purified or isolated.

51. A nucleic acid marker suitable for detecting genomic DNA or a fragment comprising a selectable marker gene excision site, wherein a selectable marker gene comprising, consisting essentially of, or consisting of a primary approved transgene locus is deleted, and wherein the nucleic acid marker does not detect the primary approved transgene locus wherein the selectable marker gene is not deleted.

52. The nucleic acid marker of claim 51, comprising a polynucleotide of at least 18 nucleotides in length spanning the selectable marker gene excision site.

53. The nucleic acid marker of claim 51, wherein the marker further comprises a detectable label.

54. The nucleic acid marker of any one of claims 51 to 53, wherein the originally approved transgene locus is Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017, MIR162, MIR604, NK603, SYN-E3272-5, 5307, DAS-40278, DP-32138, DP-33121, HCEM485, LY038, MON863, MON87403, MON87419, MON87460, mhg 0JG, mzi 098,

98140 and/or TC1507 transgene locus.

55. The nucleic acid marker of any one of claims 51 to 53, wherein the originally-approved transgene locus is a5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788,

and/or a SYHT0H2 transgene locus.

56. The nucleic acid marker of any one of claims 51 to 53, wherein the originally approved transgenic locus is a DAS-21023-5, DAS-24236-5, COT102, LL cotton 25, MON15985, MON88701, and/or MON88913 transgenic locus.

57. The nucleic acid marker of any one of claims 51 to 53, wherein the originally approved transgene locus is a GT73, HCN28, MON88302, or MS8 transgene locus.

58. A biological sample comprising plant genomic DNA or a fragment thereof, said genomic DNA or fragment comprising a selectable marker gene excision site, wherein a selectable marker gene comprising, consisting essentially of, or a segment consisting of an originally approved transgene locus is deleted.

59. The biological sample of claim 58, wherein the originally-approved transgene locus is Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017, MIR162, MIR604, NK603, SYN-E3272-5, 5307, DAS-40278, DP-33121, HCEM485, MON863, MON87403, MON87419, MON87460, MZHG0JG, mzi 098, mzi r 88017,

98140 and/or TC1507 transgene locus.

60. The biological sample of claim 58, wherein the originally approved transgene locus is A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788,

And/or a SYHT0H2 transgene locus.

61. The biological sample of claim 58, wherein the originally-approved transgenic locus is a DAS-21023-5, DAS-24236-5, COT102, LL cotton 25, MON15985, MON88701, and/or MON88913 transgenic locus.

62. The biological sample of claim 58, wherein the originally approved transgene locus is the GT73, HCN28, MON88302, or MS8 transgene locus.

63. A method of identifying a plant of any one of claims 22 to 25, a DNA of any one of claims 45 to 50 or a biological sample of any one of claims 58 to 62, the method comprising detecting a polynucleotide comprising a selectable marker gene excision site with a nucleic acid detection assay, wherein a selectable marker gene comprising, consisting essentially of, or consisting of a segment of a originally approved transgene locus is deleted.

64. The method of claim 63, wherein the detection assay does not detect an originally approved transgene locus in which the selectable marker gene is not deleted.

65. The method of claim 63, wherein the detection assay comprises contacting the biological sample with a nucleic acid marker of any one of claims 51 to 57.

66. A method of enhancing the function of an approved transgenic locus, the method comprising deleting a segment of the original approved transgenic locus with one or more gene editing molecules, the segment comprising, consisting essentially of, or consisting of:

Replication of the transgene;

replication of the transgenic element; and/or

A fragment of the transgene;

optionally, wherein the replication and/or fragment of the transgenic element is a promoter or a replication and/or fragment of a polyadenylation signal.

67. The method of claim 66, wherein the enhanced function is compared to the original approved transgene locus without the deletion.

68. The method of claim 66, wherein the enhanced function comprises reduced silencing of a complete transgene comprising the deleted approved transgene locus.

69. The method of claim 66, wherein the enhanced function comprises increased expression of a complete transgene comprising the deleted approved transgene locus.

70. The method of any one of claims 66-69, wherein the originally-approved transgenic locus comprises Bt11, DAS-59122-7, DP-4114, GA21, MON810 in the genome of a transgenic maize plant、MON87411、MON87427、MON88017、MON89034、MIR162、MIR604、NK603、SYN-E3272-5、5307、DAS-40278、DP-32138、DP-33121、HCEM485、LY038、MON863、MON87403、MON87403、MON87419、MON87460、MZHG0JG、MZIR098、

98140 and/or TC1507 transgene locus.

71. The method of any one of claims 66-69, wherein the originally-approved transgenic locus comprises a5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788,

And/or a SYHT0H2 transgene locus.

72. The method of any one of claims 66 to 69, wherein the originally-approved transgenic locus comprises a DAS-21023-5, DAS-24236-5, COT102, LL cotton 25, MON15985, MON88701, and/or MON88913 transgenic locus in the genome of a transgenic cotton plant.

73. The method of any one of claims 66 to 69, the originally approved transgenic locus comprising a GT73, HCN28, MON88302, or MS8 transgenic locus in the genome of a transgenic canola plant.

74. The method of claim 66, wherein the crop plant is a maize plant, wherein the originally-approved transgenic locus is the MIR162 transgenic locus of the maize plant, and wherein a transgenic element comprising a maize polyubiquitin promoter operably linked to DNA encoding phosphomannose isomerase (PMI) in the MIR162 transgenic locus of the maize plant is deleted.

75. The method of claim 66, wherein the crop plant is a maize plant, wherein the originally approved transgenic locus is a 1507 transgenic locus of the maize plant, and wherein a fragment of the 1507 transgenic locus of the maize plant comprising the PAT gene, a fragment of the maize ubiquitin promoter, a fragment of the agrobacterium mannopine synthase polyadenylation signal, and/or a fragment of the transgenic element of the truncated cry1F encoding fragment is deleted.

76. The method of claim 66, wherein the crop plant is a maize plant, wherein the approved transgenic locus is the MIR604 transgenic locus of a maize plant, and wherein a transgenic element comprising a NOS polyadenylation signal operably linked to DNA encoding phosphomannose isomerase (PMI) in the MIR604 transgenic locus of the maize plant is deleted.

77. The method of claim 66, wherein the crop plant is a maize plant, wherein the approved transgene locus is a GA21 transgene locus of the maize plant, and wherein one or more copies of the transgene encoding EPSPS and/or the fragment of the transgene encoding EPSPS in the GA21 transgene locus of the maize plant are deleted.

78. The method of any one of claims 66-77, wherein the approved transgene locus is further modified by deleting, consisting essentially of, or consisting of a selectable marker gene comprising the original approved transgene locus.

79. An elite crop plant comprising a modification of an originally approved transgene locus, wherein the modification comprises a deletion of a segment comprising, consisting essentially of, or consisting of:

Replication of the transgene;

replication of the transgenic element; and/or

A fragment of the transgene;

optionally, wherein the replication and/or fragment of the transgene is a promoter or a replication and/or fragment of a polyadenylation signal.

80. The elite crop plant according to claim 79, wherein the modification of the approved transgene locus exhibits enhanced function compared to the original version of the approved transgene locus.

81. The elite crop plant according to claim 80, wherein the enhanced function comprises reduced silencing of a complete transgene comprising the deleted approved transgene locus.

82. The elite crop plant according to claim 80, wherein the enhanced function comprises increased expression of a complete transgene comprising the deleted approved transgene locus.

83. The elite crop plant of any one of claims 79 to 82, wherein the originally approved transgene locus comprises Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017, MON89034, MIR162, MIR604, NK603, SYN-E3272-5, 5307, DAS-40278, DP-32138, DP-33121, HCEM485, LY038, MON863, MON87403, MON87419, MON87460, MZHG0JG, MZIR098 in the genome of the transgenic maize plant,

98140 and/or TC1507 transgene locus.

84. The elite crop plant of any one of claims 79 to 82, wherein the originally approved transgene locus comprises a5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788,

and/or a SYHT0H2 transgene locus.

85. The elite crop plant of any one of claims 79 to 82, wherein the originally approved transgenic locus comprises a DAS-21023-5, DAS-24236-5, COT102, LL cotton 25, MON15985, MON88701, and/or MON88913 transgenic locus in the genome of a transgenic cotton plant.

86. The elite crop plant according to any one of claims 79 to 82, the originally approved transgene locus comprises a GT73, HCN28, MON88302 or MS8 transgene locus in the genome of a transgenic canola plant.

87. The elite crop plant according to claim 79, wherein the crop plant is a maize plant, wherein the originally approved transgene locus is the MIR162 transgene locus of the maize plant, and wherein the modification comprises a deletion of a transgene element comprising the maize polyubiquitin promoter operably linked to DNA encoding phosphomannose isomerase (PMI) in the MIR162 transgene locus of the maize plant.

88. The elite crop plant according to claim 79, wherein the crop plant is a maize plant, wherein the originally approved transgene locus is a 1507 transgene locus of the maize plant, and wherein the modification comprises a deletion of: a fragment of the 1507 transgenic locus of the maize plant comprising the PAT gene, a fragment of the maize ubiquitin promoter, a fragment of the agrobacterium mannopine synthase polyadenylation signal and/or a fragment of the transgenic element of the truncated cry1F encoding fragment.

89. The elite crop plant according to claim 79, wherein the crop plant is a maize plant, wherein the approved transgene locus is the MIR604 transgene locus of the maize plant, and wherein the modification comprises a deletion of a transgene element comprising the NOS polyadenylation signal operably linked to DNA encoding phosphomannose isomerase (PMI) in the MIR604 transgene locus of the maize plant.

90. The elite crop plant according to claim 79, wherein the crop plant is a maize plant, wherein the approved transgene locus is a GA21 transgene locus of the maize plant, and wherein the modification comprises a deletion of one or more copies of a transgene encoding EPSPS and/or a deletion of a fragment of a transgene encoding EPSPS in a GA21 transgene locus of the maize plant.

91. The elite crop plant according to any one of claims 79 to 90, wherein the modification of the approved transgene locus further comprises a deletion of a segment comprising, consisting essentially of, or consisting of a selectable marker gene of the original approved transgene locus.

92. An edited crop plant genome comprising the genome of a crop plant of any one of claims 79-91.

93. A method of obtaining the elite crop plant according to any one of claims 79 to 91, comprising the steps of: (a) Obtaining a modified crop plant comprising an originally approved transgene locus, the modification comprising a deletion of a segment of the originally approved transgene locus of any one of claims 79 to 91, wherein the plant does not comprise the germplasm of the elite crop plant; and (b) introgressing the modified transgenic locus into the germplasm of the elite crop plant.

94. The method of claim 93, wherein the introgression comprises: (i) Crossing the crop plant of (a) with a plant comprising elite crop germplasm but lacking the modified transgene locus; (ii) Selecting a progeny plant comprising the modified transgenic locus; (iii) Backcrossing the progeny plant with a plant comprising the elite crop germplasm but lacking the modified transgene locus; and (iv) selecting a progeny plant comprising the modified transgenic locus.

95. A method of obtaining a population of large inbred seeds for commercial seed production, the method comprising selfing the elite crop plant according to any one of claims 79 to 91 and harvesting seed from the selfed elite crop plant.

96. A method of obtaining hybrid seed, the method comprising crossing a first plant comprising the edited crop plant genome of claim 92 with a second plant and harvesting seed from the crossing.

97. The method of claim 96, wherein the first plant and the second plant are in different sets of heterosis.

98. The method of claim 96 or 97, wherein the first or second plant is a pollen receptor that has become male sterile.

99. The method of claim 98, wherein the plant is rendered male sterile by emasculation, cytoplasmic male sterility, chemical crossing agents or systems, transgenes, and/or mutations in endogenous plant genes.

100. The method of any one of claims 96-99, further comprising the step of sowing the hybrid seed.