Patent Application
20230265445
The sequence listing contained in the file named “10083WO1_ST25.txt,” which is 492,562 bytes as measured in the Windows operating system, and which was created on Jul. 17, 2021 and electronically filed via EFS-Web on Jul. 26, 2021, is incorporated herein by reference in its entirety.
Transgenes which are placed into different positions in the plant genome through non-site specific integration can exhibit different levels of expression (Weising et al., 1988, Ann. Rev. Genet. 22:421-477). Such transgene insertion sites can also contain various undesirable rearrangements of the foreign DNA elements that include deletions and/or duplications. Furthermore, many transgene insertion sites can also comprise selectable or scoreable marker genes which in some instances are no longer required once a transgenic plant event containing the linked transgenes which confer desirable traits are selected.
Commercial transgenic plants typically comprise one or more independent insertions of transgenes at specific locations in the host plant genome that have been selected for features that include expression of the transgene(s) of interest and the transgene-conferred trait(s), absence or minimization of rearrangements, and normal Mendelian transmission of the trait(s) to progeny. Examples of selected transgenic corn, soybean, cotton, and canola plant events which confer traits such as herbicide tolerance and/or pest tolerance are disclosed in U.S. Pat. Nos. 7,323,556; 8,575,434; 6,040,497; 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 duplicated transgenes in transgene insertion sites in plant genomes involving use of site-specific recombinase systems (e.g., cre-lox) as well as for insertion of new genes into transgene insertion sites have been disclosed (Srivastava and Ow; Methods Mol Biol, 2015,1287:95-103; Dale and Ow, 1991, Proc. Natl Acad. Sci. USA 88, 10558-10562; Srivastava and Thomson, Plant Biotechnol J, 2016; 14(2):471-82). Such methods typically require incorporation of the recombination site sequences recognized by the recombinase at particular locations within the transgene.
Edited transgenic plant genomes comprising a first modified transgenic locus that comprises: (i) a first originator guide RNA recognition site (OgRRS) comprising a protospacer adjacent motif (PAM) site operably linked to a guide RNA hybridization site, wherein the OgRRS is located in non-transgenic plant genomic DNA of a first DNA junction polynucleotide of the first modified transgenic locus; and (ii) a first cognate guide RNA recognition site (CgRRS) comprising a protospacer adjacent motif (PAM) site operably linked to a guide RNA hybridization site located in a second DNA junction polynucleotide of the first modified transgenic locus, wherein the CgRRS is absent from transgenic plant genomes comprising a first original transgenic locus that is unmodified and wherein the OgRRS and the CgRRS can hybridize to one first guide RNA (gRNA) are provided.
Transgenic plant cells, transgenic plants, and transgenic plant pats including a seed, leaf, tuber, stem, root, or boll that comprise the edited transgenic plant genome are also provided.
Methods of obtaining a plant breeding line comprising (a) crossing two transgenic plants comprising the aforementioned edited transgenic genomes comprising an OgRRS and a CgRRS, wherein a first plant comprising the first modified transgenic locus is crossed to a second plant comprising the second modified transgenic locus; and (b) selecting a progeny plant comprising the first and second modified transgenic locus from the cross are provided. Methods for obtaining a bulked population of inbred seed for commercial seed production comprising selfing the transgenic plants comprising the aforementioned edited transgenic genomes comprising an OgRRS and a CgRRS and harvesting seed from the selfed plants are also provided. Methods of obtaining hybrid crop seed comprising crossing a first crop plant comprising the transgenic plants comprising the aforementioned edited transgenic genomes comprising an OgRRS and a CgRRS to a second crop plant and harvesting seed from the cross are provided.
DNA comprising a cognate guide RNA recognition site (CgRRS) and at least 10 bp of transgenic DNA or non-transgenic plant genomic DNA flanking the CgRRS, wherein the transgenic DNA or non-transgenic plant genomic DNA comprise transgenic or non-transgenic plant genomic DNA sequences of the second DNA junction polynucleotide of the first modified transgenic locus is provided. Processed transgenic plant products containing the aforementioned DNA and biological samples containing the aforementioned DNA are also provided.
Nucleic acid markers adapted for detection of genomic DNA or fragments thereof comprising a cognate guide RNA recognition site (CgRRS) in, adjacent to, or operably linked to a DNA junction polynucleotide of a modified transgenic locus are provided.
Methods of detecting the edited transgenic plant genomes comprising the step of detecting the presence of a polynucleotide comprising one or more of the CgRRS are provided.
Methods of obtaining an edited transgenic plant genome comprising a modified transgenic locus comprising the step of introducing a cognate guide RNA recognition site (CgRRS) in a DNA junction polynucleotide of an original transgenic locus, wherein the CgRRS is in, adjacent to, or operably linked to a DNA junction polynucleotide of the modified transgenic locus, are provided.
Methods of excising a modified transgenic locus from an edited transgenic plant genome comprising the steps of: (a) contacting any of the aforementioned edited transgenic plant genome(s) with: (i) an RNA dependent DNA endonuclease (RdDe); and (ii) a guide RNA (gRNA) capable of hybridizing to the guide RNA hybridization site of the first OgRRS and the first CgRRS; wherein the RdDe recognizes a OgRRS/gRNA and a CgRRS/gRNA hybridization complex; and, (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant wherein the modified transgenic locus flanked by the first OgRRS and the first CgRRS has been excised are provided.
Methods for obtaining inbred transgenic plant germplasm containing different transgenic traits comprising: (a) introgressing at least a first transgenic locus and a second transgenic locus into inbred germplasm to obtain a donor inbred parent plant line comprising the first and second transgenic loci, wherein a first OgRRS and a first CgRRS are operably linked to both DNA junction polynucleotides of at least the first transgenic locus and optionally wherein a second OgRRS and a second CgRRS are operably linked to the second transgenic locus; (b) contacting the transgenic plant genome of the donor inbred parent plant line with: (i) an RNA dependent DNA endonuclease (RdDe); and (ii) a guide RNA (gRNA) capable of hybridizing to the guide RNA hybridization site of the first OgRRS and the second CgRRS; wherein the RdDe recognizes a first OgRRS/gRNA and a first CgRRS/gRNA hybridization complex; and (c) selecting a transgenic plant cell, transgenic plant part, or transgenic plant comprising an edited transgenic plant genome in the inbred germplasm, wherein the first transgenic locus has been excised and the second transgenic locus is present in the inbred germplasm are provided.
Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5′ to 3′ direction. Nucleic acid sequences may be provided as DNA or as RNA, as specified; disclosure of one necessarily defines the other, as well as necessarily defines the exact complements, as is known to one of ordinary skill in the art.
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 means a value or range of values which would be understood as an equivalent of a stated value and can be greater or lesser than the value or range of values stated by 10 percent. Each value or range of values preceded by the term “about” is also intended to encompass the embodiment of the stated absolute value or range of values.
The phrase “allelic variant” as used herein refers to a polynucleotide or polypeptide sequence variant that occurs in a different strain, variety, or isolate of a given organism.
The term “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
As used herein, the phrase “approved transgenic locus” is a genetically modified plant event which has been authorized, approved, and/or de-regulated for any one of field testing, cultivation, human consumption, animal consumption, and/or import by a governmental body. Illustrative and non-limiting examples of governmental bodies which provide such approvals include the Ministry of Agriculture of Argentina, Food Standards Australia New Zealand, National Biosafety Technical Committee (CTNBio) of Brazil, Canadian Food Inspection Agency, China Ministry of Agriculture Biosafety Network, European Food Safety Authority, US Department of Agriculture, US Department of Environmental Protection, and US Food and Drug Administration.
The term “backcross”, as used herein, refers to crossing an F1 plant or plants with one of the original parents. A backcross is used to maintain or establish the identity of one parent (species) and to incorporate a particular trait from a second parent (species). The term “backcross generation”, as used herein, refers to the offspring of a backcross.
As used herein, the phrase “biological sample” refers to either intact or non-intact (e.g. milled seed or plant tissue, chopped plant tissue, lyophilized tissue) plant tissue. It may also be an extract comprising intact or non-intact seed or plant tissue. The biological sample can comprise flour, meal, syrup, oil, starch, and cereals manufactured in whole or in part to contain crop plant by-products. In certain embodiments, the biological sample is “non-regenerable” (i.e., incapable of being regenerated into a plant or plant part). In certain embodiments, the biological sample refers to a homogenate, an extract, or any fraction thereof containing genomic DNA of the organism from which the biological sample was obtained, wherein the biological sample does not comprise living cells.
As used herein, the terms “correspond,” “corresponding,” and the like, when used in the context of an nucleotide position, mutation, and/or substitution in any given polynucleotide (e.g., an allelic variant of SEQ ID NO: 1-34) with respect to the reference polynucleotide sequence (e.g., SEQ ID NO: 1-34) all refer to the position of the polynucleotide residue in the given sequence that has identity to the residue in the reference nucleotide sequence when the given polynucleotide is aligned to the reference polynucleotide sequence using a pairwise alignment algorithm (e.g., CLUSTAL O 1.2.4 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 protein provided herein as SEQ ID NO: 46.
The term “crossing” as used herein refers to the fertilization of female plants (or gametes) by male plants (or gametes). The term “gamete” refers to the haploid reproductive cell (egg or pollen) produced in plants by meiosis from a gametophyte and involved in sexual reproduction, during which two gametes of opposite sex fuse to form a diploid zygote. The term generally includes reference to a pollen (including the sperm cell) and an ovule (including the ovum). When referring to crossing in the context of achieving the introgression of a genomic region or segment, the skilled person will understand that in order to achieve the introgression of only a part of a chromosome of one plant into the chromosome of another plant, random portions of the genomes of both parental lines recombine during the cross due to the occurrence of crossing-over events in the production of the gametes in the parent lines. Therefore, the genomes of both parents must be combined in a single cell by a cross, where after the production of gametes from the cell and their fusion in fertilization will result in an introgression event.
As used herein, the phrases “DNA junction polynucleotide” and “junction polynucleotide” refers to a polynucleotide of about 18 to about 500 base pairs in length comprised of both endogenous chromosomal DNA of the plant genome and heterologous transgenic DNA which is inserted in the plant genome. A junction polynucleotide can thus comprise about 8, 10, 20, 50, 100, 200, 250, 500, or 1000 base pairs of endogenous chromosomal DNA of the plant genome and about 8, 10, 20, 50, 100, 200, 250, 500, or 1000 base pairs of heterologous transgenic DNA which span the one end of the transgene insertion site in the plant chromosomal DNA. Transgene insertion sites in chromosomes will typically contain both a 5′ junction polynucleotide and a 3′ junction polynucleotide. In embodiments set forth herein in SEQ ID NO: 1-34, the 5′ junction polynucleotide is located at the 5′ end of the sequence and the 3′ junction polynucleotide is located at the 3′ end of the sequence. In a non-limiting and illustrative example, a 5′ junction polynucleotide of a transgenic locus is telomere proximal in a chromosome arm and the 3′ junction polynucleotide of the transgenic locus is centromere proximal in the same chromosome arm. In another non-limiting and illustrative example, a 5′ junction polynucleotide of a transgenic locus is centromere proximal in a chromosome arm and the 3′ junction polynucleotide of the transgenic locus is telomere proximal in the same chromosome arm. The junction polynucleotide which is telomere proximal and the junction polynucleotide which is centromere proximal can be determined by comparing non-transgenic genomic sequence of a sequenced non-transgenic plant genome to the non-transgenic DNA in the junction polynucleotides.
The term “donor,” as used herein in the context of a plant, refers to the plant or plant line from which the trait, transgenic event, or genomic segment originates, wherein the donor can have the trait, introgression, or genomic segment in either a heterozygous or homozygous state.
As used herein, the terms “excise” and “delete,” when used in the context of a DNA molecule, are used interchangeably to refer to the removal of a given DNA segment or element (e.g., transgene element or transgenic locus) of the DNA molecule.
As used herein, the phrase “elite crop plant” refers to a plant which has undergone breeding to provide one or more trait improvements. Elite crop plant lines include plants which are an essentially homozygous, e.g. inbred or doubled haploid. Elite crop plants can include inbred lines used as is or used as pollen donors or pollen recipients in hybrid seed production (e.g. used to produce F1 plants). Elite crop plants can include inbred lines which are selfed to produce non-hybrid cultivars or varieties or to produce (e.g., bulk up) pollen donor or recipient lines for hybrid seed production. Elite crop plants can include hybrid F1 progeny of a cross between two distinct elite inbred or doubled haploid plant lines.
As used herein, an “event,” “a transgenic event,” “a transgenic locus” and related phrases refer to an insertion of one or more transgenes at a unique site in the genome of a plant as well as to DNA fragments, plant cells, plants, and plant parts (e.g., seeds) comprising genomic DNA containing the transgene insertion. Such events typically comprise both a 5′ and a 3′ DNA junction polynucleotide and confer one or more useful traits including herbicide tolerance, insect resistance, male sterility, and the like.
As used herein, the phrases “endogenous sequence,” “endogenous gene,” “endogenous DNA,” “endogenous polynucleotide,” and the like refer to the native form of a polynucleotide, gene or polypeptide in its natural location in the organism or in the genome of an organism.
The terms “exogenous” and “heterologous” as are used synonymously herein to refer to any polynucleotide (e.g. DNA molecule) that has been inserted into a new location in the genome of a plant. Non-limiting examples of an exogenous or heterologous DNA molecule include a synthetic DNA molecule, a non-naturally occurring DNA molecule, a DNA molecule found in another species, a DNA molecule found in a different location in the same species, and/or a DNA molecule found in the same strain or isolate of a species, where the DNA molecule has been inserted into a new location in the genome of a plant.
As used herein, the term “F1” refers to any offspring of a cross between two genetically unlike individuals.
The term “gene,” as used herein, refers to a hereditary unit consisting of a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a particular characteristics or trait in an organism. The term “gene” thus includes a nucleic acid (for example, DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA, or a polypeptide or its precursor. A functional polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, pesticidal activity, ligand binding, and/or signal transduction) of the RNA or polypeptide are retained.
The term “identifying,” as used herein with respect to a plant, refers to a process of establishing the identity or distinguishing character of a plant, including exhibiting a certain trait, containing one or more transgenes, and/or containing one or more molecular markers.
The term “isolated” as used herein means having been removed from its natural environment.
As used herein, the terms “include,” “includes,” and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.
As used herein, the phrase “introduced transgene” is a transgene not present in the original transgenic locus in the genome of an initial transgenic event or in the genome of a progeny line obtained from the initial transgenic event. Examples of introduced transgenes include exogenous transgenes which are inserted in a resident original transgenic locus.
As used herein, the terms “introgression,” “introgressed,” and “introgressing” refer to both a natural and artificial process, and the resulting plants, whereby traits, genes or DNA sequences of one species, variety or cultivar are moved into the genome of another species, variety or cultivar, by crossing those species. The process may optionally be completed by backcrossing to the recurrent parent. Examples of introgression include entry or introduction of a gene, a transgene, a regulatory element, a marker, a trait, a trait locus, or a chromosomal segment from the genome of one plant into the genome of another plant.
The phrase “marker-assisted selection,” as used herein, refers to the diagnostic process of identifying, optionally followed by selecting a plant from a group of plants using the presence of a molecular marker as the diagnostic characteristic or selection criterion. The process usually involves detecting the presence of a certain nucleic acid sequence or polymorphism in the genome of a plant.
The phrase “molecular marker,” as used herein, refers to an indicator that is used in methods for visualizing differences in characteristics of nucleic acid sequences. Examples of such indicators are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), microsatellite markers (e.g. SSRs), sequence-characterized amplified region (SCAR) markers, Next Generation Sequencing (NGS) of a molecular marker, cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of the markers described herein which defines a specific genetic and chromosomal location.
As used herein the terms “native” or “natural” define a condition found in nature. A “native DNA sequence” is a DNA sequence present in nature that was produced by natural means or traditional breeding techniques but not generated by genetic engineering (e.g., using molecular biology/transformation techniques).
The term “offspring”, as used herein, refers to any progeny generation resulting from crossing, selfing, or other propagation technique.
The phrase “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. When the phrase “operably linked” is used in the context of a PAM site and a guide RNA hybridization site, it refers to a PAM site which permits cleavage of at least one strand of DNA in a polynucleotide with an RNA dependent DNA endonuclease or RNA dependent DNA nickase which recognize the PAM site when a guide RNA complementary to guide RNA hybridization site sequences adjacent to the PAM site is present. A OgRRS and its CgRRS are operably linked to junction polynucleotides when they can be recognized by a gRNA and an RdDe to provide for excision of the transgenic locus or portion thereof flanked by the junction polynucleotides.
As used herein, the term “plant” includes a whole plant and any descendant, cell, tissue, or part of a plant. The term “plant parts” include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; or a plant organ (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, and explants). A plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit. A plant cell or tissue culture may be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant. Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, or stalks. In contrast, some plant cells are not capable of being regenerated to produce plants and are referred to herein as “non-regenerable” plant cells.
The term “purified,” as used herein defines an isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment and means having been increased in purity as a result of being separated from other components of the original composition. The term “purified nucleic acid” is used herein to describe a nucleic acid sequence which has been separated from other compounds including, but not limited to polypeptides, lipids and carbohydrates.
The term “recipient”, as used herein, refers to the plant or plant line receiving the trait, transgenic event or genomic segment from a donor, and which recipient may or may not have the have trait, transgenic event or genomic segment itself either in a heterozygous or homozygous state.
As used herein the term “recurrent parent” or “recurrent plant” describes an elite line that is the recipient plant line in a cross and which will be used as the parent line for successive backcrosses to produce the final desired line.
As used herein the term “recurrent parent percentage” relates to the percentage that a backcross progeny plant is identical to the recurrent parent plant used in the backcross. The percent identity to the recurrent parent can be determined experimentally by measuring genetic markers such as SNPs and/or RFLPs or can be calculated theoretically based on a mathematical formula.
The terms “selfed,” “selfing,” and “self,” as used herein, refer to any process used to obtain progeny from the same plant or plant line as well as to plants resulting from the process. As used herein, the terms thus include any fertilization process wherein both the ovule and pollen are from the same plant or plant line and plants resulting therefrom.
Typically, the terms refer to self-pollination processes and progeny plants resulting from self-pollination.
The term “selecting”, as used herein, refers to a process of picking out a certain individual plant from a group of individuals, usually based on a certain identity, trait, characteristic, and/or molecular marker of that individual.
As used herein, the phrase “originator guide RNA recognition site” or the acronym “OgRRS” refers to an endogenous or exogenous DNA polynucleotide comprising a protospacer adjacent motif (PAM) site operably linked to a guide RNA hybridization site. In certain embodiments, an OgRRS can be located in an untransformed plant chromosome or in non-transgenic DNA of a DNA junction polynucleotide of both an original transgenic locus and a modified transgenic locus. In certain embodiments, an OgRRS can be located in transgenic DNA of a DNA junction polynucleotide of both an original transgenic locus and a modified transgenic locus. In certain embodiments, an OgRRS can be located in both transgenic DNA and non-transgenic DNA of a DNA junction polynucleotide of both an original transgenic locus and a modified transgenic locus (i.e., can span transgenic and non-transgenic DNA in a DNA junction polynucleotide).
As used herein the phrase “cognate guide RNA recognition site” or the acronym “CgRRS” refer to a DNA polynucleotide comprising a PAM site operably linked to a guide RNA hybridization site, where the CgRRS is absent from transgenic plant genomes comprising a first original transgenic locus that is unmodified and where the CgRRS and its corresponding OgRRS can hybridize to a single gRNA. A CgRRS can be located in transgenic DNA of a DNA junction polynucleotide of a modified transgenic locus, in transgenic DNA of a DNA junction polynucleotide of a modified transgenic locus, or in both transgenic and non-transgenic DNA of a modified transgenic locus (i.e., can span transgenic and non-transgenic DNA in a DNA junction polynucleotide).
As used herein, the phrase “a transgenic locus excision site” refers to the DNA which remains in the genome of a plant or in a DNA molecule (e.g., an isolated or purified DNA molecule) wherein a segment comprising, consisting essentially of, or consisting of a transgenic locus or portion thereof has been deleted. In a non-limiting and illustrative example, a transgenic locus excision site can thus comprise a contiguous segment of DNA comprising at least 10 base pairs of DNA that is telomere proximal to the deleted transgenic locus or to the deleted segment of the transgenic locus and at least 10 base pairs of DNA that is centromere proximal to the deleted transgenic locus or to the deleted segment of the transgenic locus.
As used herein, the phrase “transgene element” refers to a segment of DNA comprising, consisting essentially of, or consisting of a promoter, a 5′ UTR, an intron, a coding region, a 3′UTR, or a polyadenylation signal. Polyadenylation signals include transgene elements referred to as “terminators” (e.g., NOS, pinII, rbcs, Hsp17, TubA).
To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.
Genome editing molecules can permit introduction of targeted genetic change conferring desirable traits in a variety of crop plants (Zhang et al. Genome Biol. 2018; 19: 210; Schindele et al. FEBS Lett. 2018; 592(12):1954). Desirable traits introduced into crop plants such as maize and soybean include herbicide tolerance, improved food and/or feed characteristics, male-sterility, and drought stress tolerance. Nonetheless, full realization of the potential of genome editing methods for crop improvement will entail efficient incorporation of the targeted genetic changes in germplasm of different elite crop plants adapted for distinct growing conditions. Such elite crop plants will also desirably comprise useful transgenic loci which confer various traits including herbicide tolerance, pest resistance (e.g.; insect, nematode, fungal disease, and bacterial disease resistance), conditional male sterility systems for hybrid seed production, abiotic stress tolerance (e.g., drought tolerance), improved food and/or feed quality, and improved industrial use (e.g., biofuel). Provided herein are methods whereby targeted genetic changes are efficiently combined with desired subsets of transgenic loci in elite progeny plant lines (e.g., elite inbreds used for hybrid seed production or for inbred varietal production). Also provided are plant genomes containing modified transgenic loci which can be selectively excised with a single gRNA molecule. Such modified transgenic loci comprise an originator guide RNA recognition site (OgRRS) which is identified in non-transgenic DNA of a first junction polynucleotide of the transgenic locus and cognate guide RNA recognition site (CgRRS) which is introduced (e.g., by genome editing methods) into a second junction polynucleotide of the transgenic locus and which can hybridize to the same gRNA as the OgRRS, thereby permitting excision of the modified transgenic locus or portion thereof with a single guide RNA. Use of a single guide RNA molecule rather than two guide RNA molecules to excise a transgenic locus can be advantageous. In certain embodiments, the frequency of excision of the modified transgenic locus or a portion thereof comprising the OgRRS and CgRRS in the transgenic plant cells, calli, transgenic plant parts, or transgenic plants subjected to treatment with the same guide RNA and an RdDe is greater than the frequency of excision of the modified transgenic locus or a portion thereof in control transgenic plant cells, calli, transgenic plant parts, or transgenic plants treated with two control gRNAs (e.g., two distinct gRNAs that hybridize to DNA flanking all or most of the modified transgenic locus or a portion thereof) and an RdDe. Such frequencies of excision can be determined by treating equivalent numbers of plant cells, calli, plant parts, or plants with either the single gRNA that hybridizes to the OgRRS and CgRRS plus an RdDe or with the two control gRNAs plus an RdDe and determining the number of selected plant cells, calli, plant parts, or plants where the transgenic locus or portion thereof was excised. In certain embodiments, a portion of the modified transgenic locus is excised when the OgRRS, the CgRRS, or both the OgRRS are located within transgenic DNA of the junction sequences. Since the CGRRS and/or OgRRS are typically located in or near the DNA junction polynucleotides of the transgenic locus, portions of a transgenic locus that are excised typically comprise all or the bulk of the transgene expression cassettes (e.g., promoters which are operably linked to genes which confer traits of interest and/or a selectable phenotype). In certain embodiments, an originator guide RNA recognition site (OgRRS) comprises endogenous DNA found in untransformed plants and in endogenous non-transgenic DNA of junction polynucleotides of transgenic plants containing a modified or unmodified transgenic locus. In certain embodiments, an originator guide RNA recognition site (OgRRS) comprises exogenous transgenic DNA of junction polynucleotides of transgenic plants containing a modified or unmodified transgenic locus. The OgRRS located in non-transgenic DNA, transgenic DNA, or a combination thereof in a first DNA junction polynucleotide is used to design a related cognate guide RNA recognition site (CgRRS) which is introduced (e.g., by genome editing methods) into the second junction polynucleotide of the transgenic locus. A CgRRS is thus present in junction polynucleotides of modified transgenic loci provided herein and is absent from endogenous DNA found in untransformed plants and absent from endogenous non-transgenic DNA found in junction sequences of transgenic plants containing an unmodified transgenic locus. A CgRRS is also absent from a combination of non-transgenic and transgenic DNA found in junction sequences of transgenic plants containing an unmodified transgenic locus. Also provided are unique transgenic locus excision sites created by excision of such modified transgenic loci, DNA molecules comprising the modified transgenic loci, unique transgenic locus excision sites and/or plants comprising the same, biological samples containing the DNA, nucleic acid markers adapted for detecting the DNA molecules, and related methods of identifying the elite crop plants comprising unique transgenic locus excision sites.
Further provided herein are improvements of pre-existing transgenic loci in plant genomes by directed insertion, deletion, and/or substitution of DNA within or adjacent to such insertions as well as methods for effecting and using such improvements. In certain embodiments, improved transgenic loci provided here are characterized by polynucleotide sequences that can facilitate as necessary the removal of the transgenic loci from the genome. Useful applications of such improved transgenic loci and related methods of making include targeted excision of a given transgenic locus or portions thereof in certain breeding lines to facilitate recovery of germplasm with subsets of transgenic traits tailored for specific geographic locations and/or grower preferences. Other useful applications of such improved transgenic loci and related methods of making include removal of transgenic traits from certain breeding lines when it is desirable to replace the trait in the breeding line without disrupting other transgenic loci and/or non-transgenic loci. In certain embodiments, the improved transgenic loci can provide for insertion of new transgenes that confer the replacement or other desirable trait at the genomic location of the improved transgenic locus.
Methods provided herein can be used to excise any transgenic locus where the first and second junction sequences comprising the endogenous non-transgenic genomic DNA and the heterologous transgenic DNA which are joined at the site of transgene insertion in the plant genome are known or have been determined. In certain embodiments provided herein, transgenic loci can be removed from crop plant lines to obtain crop plant lines with tailored combinations of transgenic loci and optionally targeted genetic changes. Such first and second junction sequences are readily identified in new transgenic events by inverse PCR techniques using primers which are complementary the inserted transgenic sequences. In certain embodiments, the first and second junction sequences of transgenic loci are published. Examples of transgenic loci which can be improved and used in the methods provided herein include the maize, soybean, cotton, and canola transgenic loci set forth in Tables 1, 2, 3, and 4, respectively. Transgenic junction sequences for certain events are also depicted in the drawings. Such transgenic loci set forth in Tables 1-4 are found in crop plants which have in some instances been cultivated, been placed in commerce, and/or have been described in a variety of publications by various governmental bodies. Databases which have compiled descriptions of approved transgenic loci including the loci set forth in Tables 1-4 include the International Service for the Acquisition of Agri-biotech Applications (ISAAA) database (available on the world wide web internet site “isaaa.org/gmapprovaldatabase/event”), the GenBit LLC database (available on the world wide web internet site “genbitgroup.com/en/gmo/gmodatabase”), and the Biosafety Clearing-House (BCH) database (available on the http internet site “_bch.cbd.int/database/organisms”).
1Traits: IR = Insect Resistance; HT = Herbicide Tolerance; AR = Antibiotic Resistance; MU = mannose utilization; BF = Biofuel; MS = Male Sterility; MSR = Male Sterility Restoration; Q = Food and/or Feed Quality; AST = Abiotic Stress Tolerance; YG = Yield/Growth.
2Each US Patent or Patent Application Publication is incorporated herein by reference in its entirety.
3A single transgene confers vegetative tolerance to glyphosate and exhibits glyphosate-induced male sterility.
4Resistance to coleopteran and lepidopteran insect pests.
1Traits: IR = Insect Resistance; HT = Herbicide Tolerance; AR = Antibiotic Resistance; MU = mannose utilization; BF = Biofuel; MS = Male Sterility.
2Each US Patent or Patent Application Publication is incorporated herein by reference in its entirety.
3ATCC is the American Type Culture Collection, 10801 University Boulevard Manassas, VA 20110 USA (for “PTA-XXXXX” deposits).
4NCIMB is the National Collection of Industrial, Food and Marine Bacteria, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB9YA, Scotland.
5HT to 2,4-D; glyphosate, and glufosinate; also refered to as pDAB8264.44.06.1.
6Independent IR/HT and HT events combined by breeding. IR/HT event (Cry1F, Cry1Ac synpro (Cry1Ac), and PAT) is DAS81419-2, deposited with ATCC under PTA-12006, also referred to as DAS81419-2.
7 Elk Mound Seed, 308 Railroad Street Elk Mound, WI, USA 54739.
8HT to dicamba.
9HT to both glyphosate and isoxaflutole herbicides.
10HT to glufosinate and mesotrione herbicides.
1 Traits: IR = Insect Resistance; HT = Herbicide Tolerance; AR = Antibiotic Resistance; SM = Screenable Marker
2 Both cry1Ac cotton event 3006-210-23 and cry1F cotton event 281-24-236 described in U.S. Pat. No. 7,179,965; seed comprising both events deposited with ATCC as PTA-6233.
3 Contains both the MON531 chimeric Cry1A and MON15985X Cry2Ab insertions.
4Tolerance to dicamba and glufosinate herbicides.
1 Traits: HT = Herbicide Tolerance; MS = Male Sterility
Sequences of the junction polynucleotides as well as the transgenic insert(s) of certain transgenic loci which can be improved by the methods provided herein are set forth in Tables 1-4 (e.g., SEQ ID NO: 1-34), the patent references set forth therein and incorporated herein by reference in their entireties, and elsewhere in this disclosure. The locations of the 5′ and 3′ junction polynucleotides of certain maize and soybean transgenic loci in Tables 1 and 2 are provided in Table 5. Such 5′ junction polynucleotides span the junction of the 5′ plant genomic flank nucleotides and the transgenic insert nucleotides of the indicated transgenic events (i.e., transgenic loci) in Table 5. Such 3′ junction polynucleotides span the junction of the transgenic insert nucleotides and the 3′ plant genomic flank nucleotides of the indicated transgenic events (i.e., transgenic loci). In certain embodiments provided herein, the transgenic loci set forth in Tables 1-4 (e.g., SEQ ID NO: 1-34) are referred to as “original transgenic loci.” Allelic or other variant sequences corresponding to the sequences set forth in Tables 1-4 (e.g., SEQ ID NO: 1-34), the patent references set forth therein and incorporated herein by reference in their entireties, and elsewhere in this disclosure which may be present in certain variant transgenic plant loci can also be improved by identifying sequences in the variants that correspond to the sequences of SEQ ID NO: 1-34 by performing a pairwise alignment (e.g., using CLUSTAL O 1.2.4 with default parameters) and making corresponding changes in the allelic or other variant sequences. Such allelic or other variant sequences include sequences having at least 85%, 90%, 95%, 98%, or 99% sequence identity across the entire length or at least 20, 40, 100, 500, 1,000, 2,000, 4,000, 8,000, 10,000, or 12,000 nucleotides of the sequences set forth in Tables 1-4 (e.g., SEQ ID NO: 1-34), the patent references set forth therein and incorporated herein by reference in their entireties, and elsewhere in this disclosure. Also provided are plants, plant parts including seeds, genomic DNA, and/or DNA obtained from plants set forth in Tables 1-4 which comprise one or more modifications (e.g., via insertion of a CgRRS in a junction polynucleotide sequence) which provide for their excision as well as transgenic loci excision sites wherein a segment comprising, consisting essentially of, or consisting of a transgenic locus or portion thereof is deleted. In certain embodiments, the transgenic loci set forth in Tables 1-4 and SEQ ID NO: 1-34 are further modified by deletion of a segment of DNA comprising, consisting essentially of, or consisting of a selectable marker gene and/or non-essential DNA. Also provided herein are methods of detecting plants, genomic DNA, and/or DNA obtained from plants set forth in Tables 1-4 comprising a CgRRS, deletions of selectable marker genes, deletions of non-essential DNA, or a transgenic locus excision site. A first junction polynucleotide of a transgenic locus can comprise either one of the junction polynucleotides found at the 5′ end or the 3′ end of any one of the sequences set forth in SEQ ID NO: 1-34, allelic variants thereof, or other variants thereof. An OgRRS can be found within non-transgenic DNA, transgenic DNA, or a combination thereof of either one of the junction polynucleotides of any one of SEQ ID NO: 1-34, allelic variants thereof, or other variants thereof. A second junction polynucleotide of a transgenic locus can comprise either one of the junction polynucleotides found at the 5′ or 3′ end of any one of the sequences set forth in SEQ ID NO: 1-34, allelic variants thereof, or other variants thereof. A CgRRS can be introduced within transgenic DNA, non-transgenic DNA, or a combination thereof of either one of the junction polynucleotides of any one of SEQ ID NO: 1-34, allelic variants thereof, or other variants thereof. In certain embodiments, the OgRRS is found in transgenic DNA, non-transgenic DNA, or a combination thereof in the 5′ junction polynucleotide of a transgenic locus of any one of SEQ ID NO: 1-34, allelic variants thereof, or other variants thereof and the corresponding CgRRS is introduced into the transgenic DNA, non-transgenic DNA, or a combination thereof in the 3′ junction polynucleotide of the transgenic locus of SEQ ID NO: 1-34, allelic variants thereof, or other variants thereof. In other embodiments, the OgRRS is found in non-transgenic DNA of the 3′ junction polynucleotide of transgenic locus of any one of SEQ ID NO: 1-34, allelic variants thereof, or other variants thereof and the corresponding CgRRS is introduced into the transgenic or non-transgenic DNA of the 5′ junction polynucleotide of the transgenic locus of SEQ ID NO: 1-34, allelic variants thereof, or other variants thereof.
In certain embodiments, the CgRRS is comprised in whole or in part of an exogenous DNA molecule that is introduced into a DNA junction polynucleotide by genome editing. In certain embodiments, the guide RNA hybridization site of the CgRRS is operably linked to a pre-existing PAM site in the transgenic DNA or non-transgenic DNA of the transgenic plant genome. In other embodiments, the guide RNA hybridization site of the CgRRS is operably linked to a new PAM site that is introduced in the DNA junction polynucleotide by genome editing. A CgRRS can be located in non-transgenic plant genomic DNA of a DNA junction polynucleotide of a modified transgenic locus, in transgenic DNA of a DNA junction polynucleotide of a modified transgenic locus or can span the junction of the transgenic and non-transgenic DNA of a DNA junction polynucleotide of a modified transgenic locus.
Methods provided herein can be used in a variety of breeding schemes to obtain elite crop plants comprising subsets of desired modified transgenic loci comprising an OgRRS and a CgRRS operably linked to junction polynucleotide sequences and transgenic loci excision sites where undesired transgenic loci or portions thereof have been removed (e.g., by use of the OgRRS and a CgRRS). Such methods are useful at least insofar as they allow for production of distinct useful donor plant lines each having unique sets of modified transgenic loci and, in some instances, targeted genetic changes that are tailored for distinct geographies and/or product offerings. In an illustrative and non-limiting example, a different product lines comprising transgenic loci conferring only two of three types of herbicide tolerance (e.g., glyphosate, glufosinate, and dicamba) can be obtained from a single donor line comprising three distinct transgenic loci conferring resistance to all three herbicides. In certain aspects, plants comprising the subsets of undesired transgenic loci and transgenic loci excision sites can further comprise targeted genetic changes. Such elite crop plants can be inbred plant lines or can be hybrid plant lines. In certain embodiments, at least two transgenic loci (e.g., transgenic loci in Tables 1-4 or modifications thereof wherein an OgRRS and a CgRRS site is operably linked to a first and a second junction sequence and optionally a selectable marker gene and/or non-essential DNA are deleted) are introgressed into a desired donor line comprising elite crop plant germplasm and then subjected to genome editing molecules to recover plants comprising one of the two introgressed transgenic loci as well as a transgenic loci excision site introduced by excision of the other transgenic locus or portion thereof by the genome editing molecules. In certain embodiments, the genome editing molecules can be used to remove a transgenic locus and introduce targeted genetic changes in the crop plant genome. Introgression can be achieved by backcrossing plants comprising the transgenic loci to a recurrent parent comprising the desired elite germplasm and selecting progeny with the transgenic loci and recurrent parent germplasm. Such backcrosses can be repeated and/or supplemented by molecular assisted breeding techniques using SNP or other nucleic acid markers to select for recurrent parent germplasm until a desired recurrent parent percentage is obtained (e.g., at least about 95%, 96%, 97%, 98%, or 99% recurrent parent percentage). A non-limiting, illustrative depiction of a scheme for obtaining plants with both subsets of transgenic loci and the targeted genetic changes is shown in the
Hybrid plant lines comprising elite crop plant germplasm, at least one transgenic locus and at least one transgenic locus excision site, and in certain aspects, additional targeted genetic changes are also provided herein. Methods for production of such hybrid seed can comprise crossing elite crop plant lines where at least one of the pollen donor or recipient comprises at least the transgenic locus and a transgenic locus excision site and/or additional targeted genetic changes. In certain embodiments, the pollen donor and recipient will comprise germplasm of distinct heterotic groups and provide hybrid seed and plants exhibiting heterosis. In certain embodiments, the pollen donor and recipient can each comprise a distinct transgenic locus which confers either a distinct trait (e.g., herbicide tolerance or insect resistance), a different type of trait (e.g., tolerance to distinct herbicides or to distinct insects such as coleopteran or lepidopteran insects), or a different mode-of-action for the same trait (e.g., resistance to coleopteran insects by two distinct modes-of-action or resistance to lepidopteran insects by two distinct modes-of-action). In certain embodiments, the pollen recipient will be rendered male sterile or conditionally male sterile. Methods for inducing male sterility or conditional male sterility include emasculation (e.g., detasseling), cytoplasmic male sterility, chemical hybridizing agents or systems, a transgenes or transgene systems, and/or mutation(s) in one or more endogenous plant genes. Descriptions of various male sterility systems that can be adapted for use with the elite crop plants provided herein are described in Wan et al. Molecular Plant; 12, 3, (2019):321-342 as well as in U.S. Pat. No. 8,618,358; US 20130031674; and US 2003188347.
In certain embodiments, it will be desirable to use genome editing molecules to make modified transgenic loci by introducing a CgRRS into the transgenic loci, to excise modified transgenic loci comprising an OgRRS and a CgRRS, and/or to make targeted genetic changes in elite crop plant or other germplasm. Techniques for effecting genome editing in crop plants (e.g., maize,) include use of morphogenic factors such as Wuschel (WUS), Ovule Development Protein (ODP), and/or Babyboom (BBM) which can improve the efficiency of recovering plants with desired genome edits. In some embodiments, the morphogenic factor comprises WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5, WOX9, BBM2, BMN2, BMN3, and/or ODP2. In certain embodiments, compositions and methods for using WUS, BBM, and/or ODP, as well as other techniques which can be adapted for effecting genome edits in elite crop plant and other germplasm, are set forth in US 20030082813, US 20080134353, US 20090328252, US 20100100981, US 20110165679, US 20140157453, US 20140173775, and US 20170240911, which are each incorporated by reference in their entireties. In certain embodiments, the genome edits can be effected in regenerable plant parts (e.g., plant embryos) of elite crop plants by transient provision of gene editing molecules or polynucleotides encoding the same and do not necessarily require incorporating a selectable marker gene into the plant genome (e.g., US 20160208271 and US 20180273960, both incorporated herein by reference in their entireties; Svitashev et al. Nat Commun. 2016; 7:13274).
In certain embodiments, edited transgenic plant genomes, transgenic plant cells, parts, or plants containing those genomes, and DNA molecules obtained therefrom, can comprise a desired subset of transgenic loci and/or comprise at least one transgenic locus excision site. In certain embodiments where a segment comprising an modified transgenic locus (e.g., a transgenic locus comprising an OgRRS in non-transgenic DNA of a 1st junction sequence and a CgRRS in a 2nd junction sequence) has been deleted by use of a gRNA and RdDe that recognize the OgRRS and the CgRRS, the transgenic locus excision site can comprise a contiguous segment of DNA comprising at least 10 base pairs of DNA that is telomere proximal to the deleted segment of the transgenic locus and at least 10 base pairs of DNA that is centromere proximal to the deleted segment of the transgenic locus wherein the transgenic DNA (i.e., the heterologous DNA) that has been inserted into the crop plant genome has been deleted. In certain embodiments where a segment comprising a transgenic locus has been deleted, the transgenic locus excision site can comprise a contiguous segment of DNA comprising at least 10 base pairs DNA that is telomere proximal to the deleted segment of the transgenic locus and at least 10 base pairs of DNA that is centromere proximal DNA to the deleted segment of the transgenic locus wherein the heterologous transgenic DNA and at least 1, 2, 5, 10, 20, 50, or more base pairs of endogenous DNA located in a 5′ junction sequence and/or in a 3′ junction sequence of the original transgenic locus that has been deleted. In such embodiments where DNA comprising the transgenic locus is deleted, a transgenic locus excision site can comprise at least 10 base pairs of DNA that is telomere proximal to the deleted segment of the transgenic locus and at least 10 base pairs of DNA that is centromere proximal to the deleted segment of the transgenic locus wherein all of the transgenic DNA is absent and either all or less than all of the endogenous DNA flanking the transgenic DNA sequences are present. In certain embodiments where a segment consisting essentially of an original transgenic locus has been deleted, the transgenic locus excision site can be a contiguous segment of at least 10 base pairs of DNA that is telomere proximal to the deleted segment of the transgenic locus and at least 10 base pairs of DNA that is centromere proximal to the deleted segment of the transgenic locus wherein less than all of the heterologous transgenic DNA that has been inserted into the crop plant genome is excised. In certain aforementioned embodiments where a segment consisting essentially of an original transgenic locus has been deleted, the transgenic locus excision site can thus contain at least 1 base pair of DNA or 1 to about 2 or 5, 8, 10, 20, or 50 base pairs of DNA comprising the telomere proximal and/or centromere proximal heterologous transgenic DNA that has been inserted into the crop plant genome. In certain embodiments where a segment consisting of an original transgenic locus has been deleted, the transgenic locus excision site can contain a contiguous segment of DNA comprising at least 10 base pairs of DNA that is telomere proximal to the deleted segment of the transgenic locus and at least 10 base pairs of DNA that is centromere proximal to the deleted segment of the transgenic locus wherein the heterologous transgenic DNA that has been inserted into the crop plant genome is deleted. In certain embodiments where DNA consisting of the transgenic locus is deleted, a transgenic locus excision site can comprise at least 10 base pairs of DNA that is telomere proximal to the deleted segment of the transgenic locus and at least 10 base pairs of DNA that is centromere proximal to the deleted segment of the transgenic locus wherein all of the heterologous transgenic DNA that has been inserted into the crop plant genome is deleted and all of the endogenous DNA flanking the heterologous sequences of the transgenic locus is present. In any of the aforementioned embodiments or in other embodiments, the continuous segment of DNA comprising the transgenic locus excision site can further comprise an insertion of 1 to about 2, 5, 10, 20, or more nucleotides between the DNA that is telomere proximal to the deleted segment of the transgenic locus and the DNA that is centromere proximal to the deleted segment of the transgenic locus. Such insertions can result either from endogenous DNA repair and/or recombination activities at the double stranded breaks introduced at the excision site and/or from deliberate insertion of an oligonucleotide. Plants, edited plant genomes, biological samples, and DNA molecules (e.g., including isolated or purified DNA molecules) comprising the transgenic loci excision sites are provided herein.
In other embodiments, a segment comprising a modified transgenic locus (e.g., a transgenic locus comprising an OgRRS in non-transgenic DNA of a 1st junction sequence and a CgRRS in a 2nd junction sequence) can be deleted with a gRNA and RdDe that recognize the OgRRS and the CgRRS and replaced with DNA comprising the endogenous non-transgenic plant genomic DNA present in the genome prior to transgene insertion. A non-limiting example of such replacements can be visualized in
In certain embodiments, modified versions of an approved transgenic locus are provided which can comprise an OgRRS and a CgRRS which are operably linked to a 1st and a 2nd junction sequences, respectively, and can optionally further comprise deletions of selectable marker genes. In their unmodified form (in certain embodiments, the “unmodified form” is the “original form,” “original transgenic locus,” etc.) many approved transgenic loci comprises at least one selectable marker gene. In a modified version, at least one selectable marker has been deleted with genome editing molecules as described elsewhere herein from the unmodified approved transgenic locus. In certain embodiments, the deletion of the selectable marker gene does not affect any other functionality of the approved transgenic locus. In certain embodiments, the selectable marker gene that is deleted confers resistance to an antibiotic, tolerance to an herbicide, or an ability to grow on a specific carbon source, for example, mannose. In certain embodiments, the selectable marker gene comprises a DNA encoding a phosphinothricin acetyl transferase (PAT), a glyphosate tolerant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS), a glyphosate oxidase (GOX), neomycin phosphotransferase (npt), a hygromycin phosphotransferase (hyg), an aminoglycoside adenyl transferase, or a phosphomannose isomerase (pmi). In certain embodiments, the modified locus does not contain a site-specific recombination system DNA recognition site, for example, in certain embodiments, the modified locus does not contain a lox or FRT site. In certain embodiments, the selectable marker gene to be deleted is flanked by operably linked protospacer adjacent motif (PAM) sites in the unmodified form of the approved transgenic locus. Thus, in certain embodiments of the modified locus, PAM sites flank the excision site of the deleted selectable marker gene. In certain embodiments, the PAM sites are recognized by an RNA dependent DNA endonuclease (RdDe); for example, a class 2 type II or class 2 type V RdDe. In certain embodiments, the deleted selectable marker gene is replaced in the modified approved transgenic locus by an introduced DNA sequence as discussed in further detail elsewhere herein. For example, in certain embodiments, the introduced DNA sequence comprises a trait expression cassette such as a trait expression cassette of another transgenic locus. In addition to the deletion of a selectable marker gene, in certain embodiments at least one copy of a repetitive sequence has also been deleted with genome editing molecules from an unmodified approved transgenic locus. In certain embodiments, deletion of the repetitive sequence enhances the functionality of the modified approved transgenic locus. In certain embodiments, the approved transgenic locus which is modified is: (i) a 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, MON87403, MON87419, MON87460, MZHG0JG, MZIR098, VCO-Ø1981-5, 98140, and/or TC1507 transgenic locus in a transgenic maize plant genome; (ii) an A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, and/or SYHT0H2 transgenic locus in a transgenic soybean plant genome; (iii) a DAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701, and/or MON88913 transgenic locus in a transgenic cotton plant genome; or (iv) a GT73, HCN28, MON88302, and/or MS8 transgenic locus in a transgenic canola plant genome. Also provided herein are plants comprising any of aforementioned modified transgenic loci.
In certain embodiments, edited transgenic plant genomes and transgenic plant cells, plant parts, or plants containing those edited genomes, comprising a modification of an original transgenic locus, where the modification comprises an OgRRS and a CgRRS which are operably linked to a 1st and a 2nd junction sequence, respectively, and optionally further comprise a deletion of a segment of the original transgenic locus. In certain embodiments, the modification comprises two or more separate deletions and/or there is a modification in two or more original transgenic plant loci. In certain embodiments, the deleted segment comprises, consists essentially of, or consists of a segment of non-essential DNA in the transgenic locus. Illustrative examples of non-essential DNA include but are not limited to synthetic cloning site sequences, duplications of transgene sequences; fragments of transgene sequences, and Agrobacterium right and/or left border sequences. In certain embodiments, the non-essential DNA is a duplication and/or fragment of a promoter sequence and/or is not the promoter sequence operably linked in the cassette to drive expression of a transgene. In certain embodiments, excision of the non-essential DNA improves a characteristic, functionality, and/or expression of a transgene of the transgenic locus or otherwise confers a recognized improvement in a transgenic plant comprising the edited transgenic plant genome. In certain embodiments, the non-essential DNA does not comprise DNA encoding a selectable marker gene. In certain embodiments of an edited transgenic plant genome, the modification comprises a deletion of the non-essential DNA and a deletion of a selectable marker gene. The modification producing the edited transgenic plant genome could occur by excising both the non-essential DNA and the selectable marker gene at the same time, e.g., in the same modification step, or the modification could occur step-wise. For example, an edited transgenic plant genome in which a selectable marker gene has previously been removed from the transgenic locus can comprise an original transgenic locus from which a non-essential DNA is further excised and vice versa. In certain embodiments, the modification comprising deletion of the non-essential DNA and deletion of the selectable marker gene comprises excising a single segment of the original transgenic locus that comprises both the non-essential DNA and the selectable marker gene. Such modification would result in one excision site in the edited transgenic genome corresponding to the deletion of both the non-essential DNA and the selectable marker gene. In certain embodiments, the modification comprising deletion of the non-essential DNA and deletion of the selectable marker gene comprises excising two or more segments of the original transgenic locus to achieve deletion of both the non-essential DNA and the selectable marker gene. Such modification would result in at least two excision sites in the edited transgenic genome corresponding to the deletion of both the non-essential DNA and the selectable marker gene. In certain embodiments of an edited transgenic plant genome, prior to excision, the segment to be deleted is flanked by operably linked protospacer adjacent motif (PAM) sites in the original or unmodified transgenic locus and/or the segment to be deleted encompasses an operably linked PAM site in the original or unmodified transgenic locus. In certain embodiments, following excision of the segment, the resulting edited transgenic plant genome comprises PAM sites flanking the deletion site in the modified transgenic locus. In certain embodiments of an edited transgenic plant genome, the modification comprises a modification of a 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, MON87403, MON87419, MON87460, MZHG0JG, MZIR098, VCO-Ø1981-5, 98140, and/or TC1507 original transgenic locus in a transgenic corn plant genome. In certain embodiments of an edited transgenic plant genome, the modification comprises a modification of an A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, and/or SYHT0H2 original transgenic locus in a transgenic soybean plant genome. In certain embodiments of an edited transgenic plant genome, the modification comprises a modification of a DAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701, and/or MON88913 original transgenic locus in a transgenic cotton plant genome. In certain embodiments of an edited transgenic plant genome, the modification comprises a modification of an GT73, HCN28, MON88302, and/or MS8 original transgenic locus in a transgenic canola plant genome.
Nucleic acid markers adapted for detecting the transgenic loci excision sites as well as methods for detecting the presence of DNA molecules comprising the transgenic loci excision sites are also provided herein. Methods and reagents (e.g., nucleic acid markers including nucleic acid probes and/or primers) for detecting plants, edited plant genomes, and biological samples containing DNA molecules comprising the transgenic loci excision sites and/or non-essential DNA deletions are also provided herein. Detection of the DNA molecules can be achieved by any combination of nucleic acid amplification (e.g., PCR amplification), hybridization, sequencing, and/or mass-spectrometry based techniques. Methods set forth for detecting junction nucleic acids in unmodified transgenic loci set forth in US 20190136331 and U.S. Pat. No. 9,738,904, both incorporated herein by reference in their entireties, can be adapted for use in detection of the nucleic acids provided herein. In certain embodiments, such detection is achieved by amplification and/or hybridization-based detection methods using a method (e.g., selective amplification primers) and/or probe (e.g., capable of selective hybridization or generation of a specific primer extension product) which specifically recognizes the target DNA molecule (e.g., transgenic locus excision site) but does not recognize DNA from an unmodified transgenic locus. In certain embodiments, the hybridization probes can comprise detectable labels (e.g., fluorescent, radioactive, epitope, and chemiluminescent labels). In certain embodiments, a single nucleotide polymorphism detection assay can be adapted for detection of the target DNA molecule (e.g., transgenic locus excision site).
In certain embodiments, improvements in a transgenic plant locus are obtained by introducing a new cognate guide RNA recognition site (CgRRS) which is operably linked to a DNA junction polynucleotide of the transgenic locus in the transgenic plant genome. Such CgRRS sites can be recognized by RdDe and a single suitable guide RNA directed to the CgRRS and the originator gRNA Recognition Site (OgRRS) to provide for cleavage within the junction polynucleotides which flank a modified transgenic locus. In certain embodiments, the CgRRS/gRNA and OgRRS/gRNA hybridization complexes are recognized by the same class of RdDe (e.g., Class 2 type II or Class 2 type V) or by the same RdDe (e.g., both the CgRRS/gRNA and OgRRS/gRNA hybridization complexes recognized by the same Cas9 or Cas 12 RdDe). Such CgRRS and OgRRS can be recognized by RdDe and suitable guide RNAs containing crRNA sufficiently complementary to the guide RNA hybridization site DNA sequences adjacent to the PAM site of the CgRRS and the OgRRS to provide for cleavage within or near the two junction polynucleotides. Suitable guide RNAs can be in the form of a single gRNA comprising a crRNA or in the form of a crRNA/tracrRNA complex. In the case of the OgRRS site, the PAM and guide RNA hybridization site are endogenous DNA polynucleotide molecules found in the plant genome. In certain embodiments where the CgRRS is introduced into the plant genome by genome editing, gRNA hybridization site polynucleotides introduced at the CgRRS are at least 17 or 18 nucleotides in length and are complementary to the crRNA of a guide RNA. In certain embodiments, the gRNA hybridization site sequence of the OgRRS and/or the CgRRS is about 17 or 18 to about 24 nucleotides in length. The gRNA hybridization site sequence of the OgRRS and the gRNA hybridization site of the CgRRS can be of different lengths or comprise different sequences so long as there is sufficient complementarity to permit hybridization by a single gRNA and recognition by a RdDe that recognizes and cleaves DNA at the gRNA/OgRRS and gRNA/CgRRS complex. In certain embodiments, the guide RNA hybridization site of the CgRRS comprise about a 17 or 18 to about 24 nucleotide sequence which is identical to the guide RNA hybridization site of the OgRRS. In other embodiments, the guide RNA hybridization site of the CgRRS comprise about a 17 or 18 to about 24 nucleotide sequence which has one, two, three, four, or five nucleotide insertions, deletions or substitutions when compared to the guide RNA hybridization site of the OgRRS. Certain CgRRS comprising a gRNA hybridization site containing has one, two, three, four, or five nucleotide insertions, deletions or substitutions when compared to the guide RNA hybridization site of the OgRRS can undergo hybridization with a gRNA which is complementary to the OgRRS gRNA hybridization site and be cleaved by certain RdDe. Examples of mismatches between gRNAs and guide RNA hybridization sites which allow for RdDe recognition and cleavage include mismatches resulting from both nucleotide insertions and deletions in the DNA which is hybridized to the gRNA (e.g., Lin et al., doi: 10.1093/nar/gku402). In certain embodiments, an operably linked PAM site is co-introduced with the gRNA hybridization site polynucleotide at the CgRRS. In certain embodiments, the gRNA hybridization site polynucleotides are introduced at a position adjacent to a resident endogenous PAM sequence in the junction polynucleotide sequence to form a CgRRS where the gRNA hybridization site polynucleotides are operably linked to the endogenous PAM site. In certain embodiments, non-limiting features of the OgRRS, CgRRS, and/or the gRNA hybridization site polynucleotides thereof include: (i) absence of significant homology or sequence identity (e.g., less than 50% sequence identity across the entire length of the OgRRS, CgRRS, and/or the gRNA hybridization site sequence) to any other endogenous or transgenic sequences present in the transgenic plant genome or in other transgenic genomes of the particular crop plant being transformed and edited (e.g., corn, soybean, cotton, canola, rice, wheat, and the like); (ii) absence of significant homology or sequence identity (e.g., less than 50% sequence identity across the entire length of the sequence) of a sequence of a first OgRRS and a first CgRRS to a second OgRRS and a second CgRRS which are operably linked to junction polynucleotides of a distinct transgenic locus; (iii) the presence of some sequence identity (e.g., about 25%, 40%, or 50% to about 60%, 70%, or 80%) between the OgRRS sequence and endogenous sequences present at the site where the CgRRS sequence is introduced; and/or (iv) optimization of the gRNA hybridization site polynucleotides for recognition by the RdDe and guide RNA when used in conjunction with a particular PAM sequence. In certain embodiments, the first and second OgRRS as well as the first and second CgRRS are recognized by the same class of RdDe (e.g., Class 2 type II or Class 2 type V) or by the same RdDe (e.g., Cas9 or Cas 12 RdDe). In certain embodiments, the first OgRRS site in a first junction polynucleotide and the CgRRS introduced in the second junction polynucleotide to permit excision of a first transgenic locus or portion thereof by a first single guide RNA and a single RdDe. Such nucleotide insertions or genome edits used to introduce CgRRS in a transgenic plant genome can be effected in the plant genome by using gene editing molecules (e.g., RdDe and guide RNAs, RNA dependent nickases and guide RNAs, Zinc Finger nucleases or nickases, or TALE nucleases or nickases) which introduce blunt double stranded breaks or staggered double stranded breaks in the DNA junction polynucleotides. In the case of DNA insertions, the genome editing molecules can also in certain embodiments further comprise a donor DNA template or other DNA template which comprises the heterologous nucleotides for insertion to form the CgRRS. Guide RNAs can be directed to the junction polynucleotides by using a pre-existing PAM site located within or adjacent to a junction polynucleotide of the transgenic locus. Non-limiting examples of such pre-existing PAM sites present in junction polynucleotides, which can be used either in conjunction with an inserted heterologous sequence to form a CgRRS or which can be used to create a double stranded break to insert or create a CgRRS, include PAM sites recognized by a Cas9 or Cas12a enzyme. Non-limiting examples where CgRRS are created in a DNA sequence are illustrated in the Examples.
Transgenic loci or portions thereof comprising OgRRS and CgRRS in a first and a second junction polynucleotides can be excised from the genomes of transgenic plants by contacting the transgenic loci with RdDe or RNA directed nickases, and a suitable guide RNA directed to the OgRRS and CgRRS. A non-limiting example where a modified transgenic locus is excised from a plant genome by use of a gRNA and an RdDe that recognizes an OgRRS/gRNA and a CgRRS/gRNA complex and introduces dsDNA breaks in both junction polynucleotides nd repaired by NHEJ is depicted in
In certain embodiments, edited transgenic plant genomes provided herein can lack one or more selectable and/or scoreable markers found in an original event (transgenic locus). Original transgenic loci (events), including those set forth in Tables 1-4 (e.g., SEQ ID NO: 1-34), the patent references set forth therein and incorporated herein by reference in their entireties, and depicted in the drawings, can contain selectable transgenes markers conferring herbicide tolerance, antibiotic resistance, or an ability to grow on a carbon source. Selectable marker transgenes which can confer herbicide tolerance include genes encoding a phosphinothricin acetyl transferase (PAT), a glyphosate tolerant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS), and a glyphosate oxidase (GOX). Selectable marker transgenes which can confer antibiotic resistance include genes encoding a neomycin phosphotransferase (npt), a hygromycin phosphotransferase, and an aminoglycoside adenyl transferase. Transgenes encoding a phosphomannose isomerase (pmi) can confer the ability to grow on mannose. Original transgenic loci (events), including those set forth in Tables 1-4 (e.g., SEQ ID NO: 1-34) and the patent references set forth therein which are incorporated herein by reference in their entireties, can contain scoreable transgenic markers which can be detected by enzymatic, histochemical, or other assays. Scoreable markers can include genes encoding beta-glucuronidase (uid) or fluorescent proteins (e.g., a GFP, RFP, or YFP). Such selectable or scoreable marker transgenes can be excised from an original transgenic locus by contacting the transgenic locus with one or more gene editing molecules which introduce double stranded breaks in the transgenic locus at the 5′ and 3′ end of the expression cassette comprising the selectable marker transgene (e.g., an RdDe and guide RNAs directed to PAM sites located at the 5′ and 3′ end of the expression cassette comprising the selectable marker transgenes) and selecting for plant cells, plant parts, or plants wherein the selectable or scoreable marker has been excised. In certain embodiments, the selectable or scoreable marker transgene can be inactivated. Inactivation can be achieved by modifications including insertion, deletion, and/or substitution of one or more nucleotides in a promoter element, 5′ or 3′ untranslated region (UTRs), intron, coding region, and/or 3′ terminator and/or polyadenylation site of the selectable marker transgene. Such modifications can inactivate the selectable or scoreable marker transgene by eliminating or reducing promoter activity, introducing a missense mutation, and/or introducing a pre-mature stop codon. In certain embodiments, the selectable and/or scoreable marker transgene can be replaced by an introduced transgene. In certain embodiments, an original transgenic locus that was contacted with gene editing molecules which introduce double stranded breaks in the transgenic locus at the 5′ and 3′ end of the expression cassette comprising the selectable marker and/or scoreable transgene can also be contacted with a suitable donor DNA template comprising an expression cassette flanked by DNA homologous to remaining DNA in the transgenic locus located 5′ and 3′ to the selectable marker excision site. In certain embodiments, a coding region of the selectable and/or scoreable marker transgene can be replaced with another coding region such that the replacement coding region is operably linked to the promoter and 3′ terminator or polyadenylation site of the selectable and/or scoreable marker transgene.
In certain embodiments, edited transgenic plant genomes provided herein can comprise additional new introduced transgenes (e.g., expression cassettes) inserted into the transgenic locus of a given event. Introduced transgenes inserted at the transgenic locus of an event subsequent to the event's original isolation can be obtained by inducing a double stranded break at a site within an original transgenic locus (e.g., with genome editing molecules including an RdDe and suitable guide RNA(s); a suitable engineered zinc-finger nuclease; a TALEN protein and the like) and providing an exogenous transgene in a donor DNA template which can be integrated at the site of the double stranded break (e.g. by homology-directed repair (HDR) or by non-homologous end-joining (NHEJ)). In certain embodiments, an OgRRS and a CgRRS located in a 1st junction polynucleotide and a 2nd junction polynucleotide, respectively, can be used to delete the transgenic locus and replace it with one or more new expression cassettes. In certain embodiments, such deletions and replacements are effected by introducing dsDNA breaks in both junction polynucleotides and providing the new expression cassettes on a donor DNA template (e.g., in
In certain embodiments, plants provided herein, including plants with one or more transgenic loci, modified transgenic loci, and/or comprising transgenic loci excision sites can further comprise one or more targeted genetic changes introduced by one or more of gene editing molecules or systems. Also provided are methods where the targeted genetic changes and one or more transgenic loci excision sites are removed from plants either in series or in parallel (e.g., as set forth in the non-limiting illustration in
Gene editing molecules of use in methods provided herein include molecules capable of introducing a double-strand break (“DSB”) or single-strand break (“SSB”) in double-stranded DNA, such as in genomic DNA or in a target gene located within the genomic DNA as well as accompanying guide RNA or donor DNA template polynucleotides. Examples of such gene editing molecules include: (a) a nuclease comprising an RNA-guided nuclease, an RNA-guided DNA endonuclease or RNA directed DNA endonuclease (RdDe), a class 1 CRISPR type nuclease system, a type II Cas nuclease, a Cas9, a nCas9 nickase, a type V Cas nuclease, a Cas12a nuclease, a nCas12a nickase, a Cas12d (CasY), a Cas12e (CasX), a Cas12b (C2c1), a Cas12c (C2c3), a Cas12i, a Cas12j, a Cas14, an engineered nuclease, a codon-optimized nuclease, a zinc-finger nuclease (ZFN) or nickase, a transcription activator-like effector nuclease (TAL-effector nuclease or TALEN) or nickase (TALE-nickase), an Argonaute, and a meganuclease or engineered meganuclease; (b) a polynucleotide encoding one or more nucleases capable of effectuating site-specific alteration (including introduction of a DSB or SSB) of a target nucleotide sequence; (c) a guide RNA (gRNA) for an RNA-guided nuclease, or a DNA encoding a gRNA for an RNA-guided nuclease; (d) donor DNA template polynucleotides; and (e) other DNA templates (dsDNA, ssDNA, or combinations thereof) suitable for insertion at a break in genomic DNA (e.g., by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ).
CRISPR-type genome editing can be adapted for use in the plant cells and methods provided herein in several ways. CRISPR elements, e.g., gene editing molecules comprising CRISPR endonucleases and CRISPR guide RNAs including single guide RNAs or guide RNAs in combination with tracrRNAs or scoutRNA, or polynucleotides encoding the same, are useful in effectuating genome editing without remnants of the CRISPR elements or selective genetic markers occurring in progeny. In certain embodiments, the CRISPR elements are provided directly to the eukaryotic cell (e.g., plant cells), systems, methods, and compositions as isolated molecules, as isolated or semi-purified products of a cell free synthetic process (e.g., in vitro translation), or as isolated or semi-purified products of in a cell-based synthetic process (e.g., such as in a bacterial or other cell lysate). In certain embodiments, genome-inserted CRISPR elements are useful in plant lines adapted for use in the methods provide herein. In certain embodiments, plants or plant cells used in the systems, methods, and compositions provided herein can comprise a transgene that expresses a CRISPR endonuclease (e.g., a Cas9, a Cpf1-type or other CRISPR endonuclease). In certain embodiments, one or more CRISPR endonucleases with unique PAM recognition sites can be used. Guide RNAs (sgRNAs or crRNAs and a tracrRNA) to form an RNA-guided endonuclease/guide RNA complex which can specifically bind sequences in the gDNA target site that are adjacent to a protospacer adjacent motif (PAM) sequence. The type of RNA-guided endonuclease typically informs the location of suitable PAM sites and design of crRNAs or sgRNAs. G-rich PAM sites, e.g., 5′-NGG are typically targeted for design of crRNAs or sgRNAs used with Cas9 proteins. Examples of PAM sequences include 5′-NGG (Streptococcus pyogenes), 5′-NNAGAA (Streptococcus thermophilus CRISPR1), 5′-NGGNG (Streptococcus thermophilus CRISPR3), 5′-NNGRRT or 5′-NNGRR (Staphylococcus aureus Cas9, SaCas9), and 5′-NNNGATT (Neisseria meningitidis). T-rich PAM sites (e.g., 5′-TTN or 5′-TTTV, where “V” is A, C, or G) are typically targeted for design of crRNAs or sgRNAs used with Cas12a proteins. In some instances, Cas12a can also recognize a 5′-CTA PAM motif. Other examples of potential Cas12a PAM sequences include TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN, ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN, GCCN, and CCGN (wherein N is defined as any nucleotide). Cpf1 endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016/0208243 A1, which is incorporated herein by reference for its disclosure of DNA encoding Cpf1 endonucleases and guide RNAs and PAM sites. Introduction of one or more of a wide variety of CRISPR guide RNAs that interact with CRISPR endonucleases integrated into a plant genome or otherwise provided to a plant is useful for genetic editing for providing desired phenotypes or traits, for trait screening, or for gene editing mediated trait introgression (e.g., for introducing a trait into a new genotype without backcrossing to a recurrent parent or with limited backcrossing to a recurrent parent). Multiple endonucleases can be provided in expression cassettes with the appropriate promoters to allow multiple genome site editing.
CRISPR technology for editing the genes of eukaryotes is disclosed in US Patent Application Publications 2016/0138008A1 and US2015/0344912A1, and in U.S. Pat. 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 RNAs and PAM sites are disclosed in US Patent Application Publication 2016/0208243 A1. Other CRISPR nucleases useful for editing genomes include Cas12b and Cas12c (see Shmakov et al. (2015) Mol. Cell, 60:385-397; Harrington et al. (2020) Molecular Cell doi:10.1016/j.molcel.2020.06.022) and CasX and CasY (see Burstein et al. (2016) Nature, doi:10.1038/nature21059; Harrington et al. (2020) Molecular Cell doi:10.1016/j.molcel.2020.06.022), or Cas12j (Pausch et al, (2020) Science 10.1126/science.abb1400). Plant RNA promoters for expressing CRISPR guide RNA and plant codon-optimized CRISPR Cas9 endonuclease are disclosed in International Patent Application PCT/US2015/018104 (published as WO 2015/131101 and claiming priority to U.S. Provisional Patent Application 61/945,700). Methods of using CRISPR technology for genome editing in plants are disclosed in US Patent Application Publications US 2015/0082478A1 and US 2015/0059010A1 and in International Patent Application PCT/US2015/038767 A1 (published as WO 2016/007347 and claiming priority to U.S. Provisional Patent Application 62/023,246). All of the patent publications referenced in this paragraph are incorporated herein by reference in their entirety. In certain embodiments, an RNA-guided endonuclease that leaves a blunt end following cleavage of the target site is used. Blunt-end cutting RNA-guided endonucleases include Cas9, Cas12c, and Cas 12h (Yan et al., 2019). In certain embodiments, an RNA-guided endonuclease that leaves a staggered single stranded DNA overhanging end following cleavage of the target site following cleavage of the target site is used. Staggered-end cutting RNA-guided endonucleases include Cas12a, Cas12b, and Cas12e.
The methods can also use sequence-specific endonucleases or sequence-specific endonucleases and guide RNAs that cleave a single DNA strand in a dsDNA target site. Such cleavage of a single DNA strand in a dsDNA target site is also referred to herein and elsewhere as “nicking” and can be affected by various “nickases” or systems that provide for nicking. Nickases that can be used include nCas9 (Cas9 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), a zinc finger nickase e.g., as disclosed in Kim et al., 2012), a TALE nickase (e.g., as disclosed in Wu et al., 2014), or a combination thereof. In certain embodiments, systems that provide for nicking can comprise a Cas nuclease (e.g., Cas9 and/or Cas12a) and guide RNA molecules that have at least one base mismatch to DNA sequences in the target editing site (Fu et al., 2019). In certain embodiments, genome modifications can be introduced into the target editing site by creating single stranded breaks (i.e., “nicks”) in genomic locations separated by no more than about 10, 20, 30, 40, 50, 60, 80, 100, 150, or 200 base pairs of DNA. In certain illustrative and non-limiting embodiments, two nickases (i.e., a CAS nuclease which introduces a single stranded DNA break including nCas9, nCas12a, Cas12i, Cas 12j, zinc finger nickases, TALE nickases, combinations thereof, and the like) or nickase systems can directed to make cuts to nearby sites separated by no more than about 10, 20, 30, 40, 50, 60, 80 or 100 base pairs of DNA. In instances where an RNA guided nickase and an RNA guide are used, the RNA guides are adjacent to PAM sequences that are sufficiently close (i.e., separated by no more than about 10, 20, 30, 40, 50, 60, 80, 100, 150, or 200 base pairs of DNA). For the purposes of gene editing, CRISPR arrays can be designed to contain one or multiple guide RNA sequences corresponding to a desired target DNA sequence; see, for example, Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308. At least 16 or 17 nucleotides of gRNA sequence are required by Cas9 for DNA cleavage to occur; for Cpf1 at least 16 nucleotides of gRNA sequence are needed to achieve detectable DNA cleavage and at least 18 nucleotides of gRNA sequence were reported necessary for efficient DNA cleavage in vitro; see Zetsche et al. (2015) Cell, 163:759-771. In practice, guide RNA sequences are generally designed to have a length of 17-24 nucleotides (frequently 19, 20, or 21 nucleotides) and exact complementarity (i.e., perfect base-pairing) to the targeted gene or nucleic acid sequence; guide RNAs having less than 100% complementarity to the target sequence can be used (e.g., a gRNA with a length of 20 nucleotides and 1-4 mismatches to the target sequence) but can increase the potential for off-target effects. The design of effective guide RNAs for use in plant genome editing is disclosed in US Patent Application Publication 2015/0082478 A1, the entire specification of which is incorporated herein by reference. More recently, efficient gene editing has been achieved using a chimeric “single guide RNA” (“sgRNA”), an engineered (synthetic) single RNA molecule that mimics a naturally occurring crRNA-tracrRNA complex and contains both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing); see, for example, Cong et al. (2013) Science, 339:819-823; Xing et al. (2014) BMC Plant Biol., 14:327-340. Chemically modified sgRNAs have been demonstrated to be effective in genome editing; see, for example, Hendel et al. (2015) Nature Biotechnol., 985-991. The design of effective gRNAs for use in plant genome editing is disclosed in US Patent Application Publication 2015/0082478 A1, the entire specification of which is incorporated herein by reference.
Genomic DNA may also be modified via base editing. Both adenine base editors (ABE) which convert A/T base pairs to G/C base pairs in genomic DNA as well as cytosine base pair editors (CBE) which effect C to T substitutions can be used in certain embodiments of the methods provided herein. In certain embodiments, useful ABE and CBE can comprise genome site specific DNA binding elements (e.g., RNA-dependent DNA binding proteins including catalytically inactive Cas9 and Cas12 proteins or Cas9 and Cas12 nickases) operably linked to adenine or cytidine deaminases and used with guide RNAs which position the protein near the nucleotide targeted for substitution. Suitable ABE and CBE disclosed in the literature (Kim, Nat Plants, 2018 March; 4(3):148-151) can be adapted for use in the methods set forth herein. In certain embodiments, a CBE can comprise a fusion between a catalytically inactive Cas9 (dCas9) RNA dependent DNA binding protein fused to a cytidine deaminase which converts cytosine (C) to uridine (U) and selected guide RNAs, thereby effecting a C to T substitution; see Komor et al. (2016) Nature, 533:420-424. In other embodiments, C to T substitutions are effected with Cas9 nickase [Cas9n(D10A)] fused to an improved cytidine deaminase and optionally a bacteriophage Mu dsDNA (double-stranded DNA) end-binding protein Gam; see Komor et al., Sci Adv. 2017 August; 3(8):eaao4774. In other embodiments, adenine base editors (ABEs) comprising an adenine deaminase fused to catalytically inactive Cas9 (dCas9) or a Cas9 D10A nickase can be used to convert A/T base pairs to G/C base pairs in genomic DNA (Gaudelli et al., (2017) Nature 551(7681):464-471.
In certain embodiments, zinc finger nucleases or zinc finger nickases can also be used in the methods provided herein. Zinc-finger nucleases are site-specific endonucleases comprising two protein domains: a DNA-binding domain, comprising a plurality of individual zinc finger repeats that each recognize between 9 and 18 base pairs, and a DNA-cleavage domain that comprises a nuclease domain (typically Fokl). The cleavage domain dimerizes in order to cleave DNA; therefore, a pair of ZFNs are required to target non-palindromic target polynucleotides. In certain embodiments, zinc finger nuclease and zinc finger nickase design methods which have been described (Urnov et al. (2010) Nature Rev. Genet., 11:636-646; Mohanta et al. (2017) Genes vol. 8, 12: 399; Ramirez et al. Nucleic Acids Res. (2012); 40(12): 5560-5568; Liu et al. (2013) Nature Communications, 4: 2565) can be adapted for use in the methods set forth herein. The zinc finger binding domains of the zinc finger nuclease or nickase provide specificity and can be engineered to specifically recognize any desired target DNA sequence. The zinc finger DNA binding domains are derived from the DNA-binding domain of a large class of eukaryotic transcription factors called zinc finger proteins (ZFPs). The DNA-binding domain of ZFPs typically contains a tandem array of at least three zinc “fingers” each recognizing a specific triplet of DNA. A number of strategies can be used to design the binding specificity of the zinc finger binding domain. One approach, termed “modular assembly”, relies on the functional autonomy of individual zinc fingers with DNA. In this approach, a given sequence is targeted by identifying zinc fingers for each component triplet in the sequence and linking them into a multifinger peptide. Several alternative strategies for designing zinc finger DNA binding domains have also been developed. These methods are designed to accommodate the ability of zinc fingers to contact neighboring fingers as well as nucleotide bases outside their target triplet. Typically, the engineered zinc finger DNA binding domain has a novel binding specificity, compared to a naturally-occurring zinc finger protein. Engineering methods include, for example, rational design and various types of selection. Rational design includes, for example, the use of databases of triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, e.g., U.S. Pat. Nos. 6,453,242 and 6,534,261, both incorporated herein by reference in their entirety. Exemplary selection methods (e.g., phage display and yeast two-hybrid systems) can be adapted for use in the methods described herein. In addition, enhancement of binding specificity for zinc finger binding domains has been described in U.S. Pat. No. 6,794,136, incorporated herein by reference in its entirety. In addition, individual zinc finger domains may be linked together using any suitable linker sequences. Examples of linker sequences are publicly known, e.g., see U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949, incorporated herein by reference in their entirety. The nucleic acid cleavage domain is non-specific and is typically a restriction endonuclease, such as Fokl. This endonuclease must dimerize to cleave DNA. Thus, cleavage by Fokl as part of a ZFN requires two adjacent and independent binding events, which must occur in both the correct orientation and with appropriate spacing to permit dimer formation. The requirement for two DNA binding events enables more specific targeting of long and potentially unique recognition sites. Fokl variants with enhanced activities have been described and can be adapted for use in the methods described herein; see, e.g., Guo et al. (2010) J. Mol. Biol., 400:96-107.
Transcription activator like effectors (TALEs) are proteins secreted by certain Xanthomonas species to modulate gene expression in host plants and to facilitate the colonization by and survival of the bacterium. TALEs act as transcription factors and modulate expression of resistance genes in the plants. Recent studies of TALEs have revealed the code linking the repetitive region of TALEs with their target DNA-binding sites. TALEs comprise a highly conserved and repetitive region consisting of tandem repeats of mostly 33 or 34 amino acid segments. The repeat monomers differ from each other mainly at amino acid positions 12 and 13. A strong correlation between unique pairs of amino acids at positions 12 and 13 and the corresponding nucleotide in the TALE-binding site has been found. The simple relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for the design of DNA binding domains of any desired specificity. TALEs can be linked to a non-specific DNA cleavage domain to prepare genome editing proteins, referred to as TAL-effector nucleases or TALENs. As in the case of ZFNs, a restriction endonuclease, such as Fokl, can be conveniently used. Methods for use of TALENs in plants have been described and can be adapted for use in the methods described herein, see Mahfouz et al. (2011) Proc. Natl. Acad. Sci. USA, 108:2623-2628; Mahfouz (2011) GM Crops, 2:99-103; and Mohanta et al. (2017) Genes vol. 8, 12: 399). TALE nickases have also been described and can be adapted for use in methods described herein (Wu et al.; Biochem Biophys Res Commun. (2014); 446(1):261-6; Luo et al; Scientific Reports 6, Article number: 20657 (2016)).
Embodiments of the donor DNA template molecule having a sequence that is integrated at the site of at least one double-strand break (DSB) in a genome include double-stranded DNA, a single-stranded DNA, a single-stranded DNA/RNA hybrid, and a double-stranded DNA/RNA hybrid. In embodiments, a donor DNA template molecule that is a double-stranded (e.g., a dsDNA or dsDNA/RNA hybrid) molecule is provided directly to the plant protoplast or plant cell in the form of a double-stranded DNA or a double-stranded DNA/RNA hybrid, or as two single-stranded DNA (ssDNA) molecules that are capable of hybridizing to form dsDNA, or as a single-stranded DNA molecule and a single-stranded RNA (ssRNA) molecule that are capable of hybridizing to form a double-stranded DNA/RNA hybrid; that is to say, the double-stranded polynucleotide molecule is not provided indirectly, for example, by expression in the cell of a dsDNA encoded by a plasmid or other vector. In various non-limiting embodiments of the method, the donor DNA template molecule that is integrated (or that has a sequence that is integrated) at the site of at least one double-strand break (DSB) in a genome is double-stranded and blunt-ended; in other embodiments the donor DNA template molecule is double-stranded and has an overhang or “sticky end” consisting of unpaired nucleotides (e.g., 1, 2, 3, 4, 5, or 6 unpaired nucleotides) at one terminus or both termini. In an embodiment, the DSB in the genome has no unpaired nucleotides at the cleavage site, and the donor DNA template molecule that is integrated (or that has a sequence that is integrated) at the site of the DSB is a blunt-ended double-stranded DNA or blunt-ended double-stranded DNA/RNA hybrid molecule, or alternatively is a single-stranded DNA or a single-stranded DNA/RNA hybrid molecule. In another embodiment, the DSB in the genome has one or more unpaired nucleotides at one or both sides of the cleavage site, and the donor DNA template molecule that is integrated (or that has a sequence that is integrated) at the site of the DSB is a double-stranded DNA or double-stranded DNA/RNA hybrid molecule with an overhang or “sticky end” consisting of unpaired nucleotides at one or both termini, or alternatively is a single-stranded DNA or a single-stranded DNA/RNA hybrid molecule; in embodiments, the donor DNA template molecule DSB is a double-stranded DNA or double-stranded DNA/RNA hybrid molecule that includes an overhang at one or at both termini, wherein the overhang consists of the same number of unpaired nucleotides as the number of unpaired nucleotides created at the site of a DSB by a nuclease that cuts in an off-set fashion (e.g., where a Cas12 nuclease effects an off-set DSB with 5-nucleotide overhangs in the genomic sequence, the donor DNA template molecule that is to be integrated (or that has a sequence that is to be integrated) at the site of the DSB is double-stranded and has 5 unpaired nucleotides at one or both termini). In certain embodiments, one or both termini of the donor DNA template molecule contain no regions of sequence homology (identity or complementarity) to genomic regions flanking the DSB; that is to say, one or both termini of the donor DNA template molecule contain no regions of sequence that is sufficiently complementary to permit hybridization to genomic regions immediately adjacent to the location of the DSB. In embodiments, the donor DNA template molecule contains no homology to the locus of the DSB, that is to say, the donor DNA template molecule contains no nucleotide sequence that is sufficiently complementary to permit hybridization to genomic regions immediately adjacent to the location of the DSB. In embodiments, the donor DNA template molecule is at least partially double-stranded and includes 2-20 base-pairs, e. g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 base-pairs; in embodiments, the donor DNA template molecule is double-stranded and blunt-ended and consists of 2-20 base-pairs, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 base-pairs; in other embodiments, the donor DNA template molecule is double-stranded and includes 2-20 base-pairs, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 base-pairs and in addition has at least one overhang or “sticky end” consisting of at least one additional, unpaired nucleotide at one or at both termini. In an embodiment, the donor DNA template molecule that is integrated (or that has a sequence that is integrated) at the site of at least one double-strand break (DSB) in a genome is a blunt-ended double-stranded DNA or a blunt-ended double-stranded DNA/RNA hybrid molecule of about 18 to about 300 base-pairs, or about 20 to about 200 base-pairs, or about 30 to about 100 base-pairs, and having at least one phosphorothioate bond between adjacent nucleotides at a 5′ end, 3′ end, or both 5′ and 3′ ends. In embodiments, the donor DNA template molecule includes single strands of at least 11, at least 18, at least 20, at least 30, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 240, at about 280, or at least 320 nucleotides. In embodiments, the donor DNA template molecule has a length of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 base-pairs if double-stranded (or nucleotides if single-stranded), or between about 2 to about 320 base-pairs if double-stranded (or nucleotides if single-stranded), or between about 2 to about 500 base-pairs if double-stranded (or nucleotides if single-stranded), or between about 5 to about 500 base-pairs if double-stranded (or nucleotides if single-stranded), or between about 5 to about 300 base-pairs if double-stranded (or nucleotides if single-stranded), or between about 11 to about 300 base-pairs if double-stranded (or nucleotides if single-stranded), or about 18 to about 300 base-pairs if double-stranded (or nucleotides if single-stranded), or between about 30 to about 100 base-pairs if double-stranded (or nucleotides if single-stranded). In embodiments, the donor DNA template molecule includes chemically modified nucleotides (see, e.g., the various modifications of internucleotide linkages, bases, and sugars described in Verma and Eckstein (1998) Annu. Rev. Biochem., 67:99-134); in embodiments, the naturally occurring phosphodiester backbone of the donor DNA template molecule is partially or completely modified with phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications, or the donor DNA template molecule includes modified nucleoside bases or modified sugars, or the donor DNA template molecule is labelled with a fluorescent moiety (e.g., fluorescein or rhodamine or a fluorescent nucleoside analogue) or other detectable label (e.g., biotin or an isotope). In another embodiment, the donor DNA template molecule contains secondary structure that provides stability or acts as an aptamer. Other related embodiments include double-stranded DNA/RNA hybrid molecules, single-stranded DNA/RNA hybrid donor molecules, and single-stranded DNA donor molecules (including single-stranded, chemically modified DNA donor molecules), which in analogous procedures are integrated (or have a sequence that is integrated) at the site of a double-strand break.
Donor DNA template molecules used in the methods provided herein include DNA molecules comprising, from 5′ to 3′, a first homology arm, a replacement DNA, and a second homology arm, wherein the homology arms containing sequences that are partially or completely homologous to genomic DNA (gDNA) sequences flanking a target site-specific endonuclease cleavage site in the gDNA. In certain embodiments, the replacement DNA can comprise an insertion, deletion, or substitution of 1 or more DNA base pairs relative to the target gDNA. In an embodiment, the donor DNA template molecule is double-stranded and perfectly base-paired through all or most of its length, with the possible exception of any unpaired nucleotides at either terminus or both termini. In another embodiment, the donor DNA template molecule is double-stranded and includes one or more non-terminal mismatches or non-terminal unpaired nucleotides within the otherwise double-stranded duplex. In an embodiment, the donor DNA template molecule that is integrated at the site of at least one double-strand break (DSB) includes between 2-20 nucleotides in one (if single-stranded) or in both strands (if double-stranded), e. g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides on one or on both strands, each of which can be base-paired to a nucleotide on the opposite strand (in the case of a perfectly base-paired double-stranded polynucleotide molecule). Such donor DNA templates can be integrated in genomic DNA containing blunt and/or staggered double stranded DNA breaks by homology-directed repair (HDR). In certain embodiments, a donor DNA template homology arm can be about 20, 50, 100, 200, 400, or 600 to about 800, or 1000 base pairs in length. In certain embodiments, a donor DNA template molecule can be delivered to a plant cell) in a circular (e.g., a plasmid or a viral vector including a geminivirus vector) or a linear DNA molecule. In certain embodiments, a circular or linear DNA molecule that is used can comprise a modified donor DNA template molecule comprising, from 5′ to 3′, a first copy of the target sequence-specific endonuclease cleavage site sequence, the first homology arm, the replacement DNA, the second homology arm, and a second copy of the target sequence-specific endonuclease cleavage site sequence. Without seeking to be limited by theory, such modified donor DNA template molecules can be cleaved by the same sequence-specific endonuclease that is used to cleave the target site gDNA of the eukaryotic cell to release a donor DNA template molecule that can participate in HDR-mediated genome modification of the target editing site in the plant cell genome. In certain embodiments, the donor DNA template can comprise a linear DNA molecule comprising, from 5′ to 3′, a cleaved target sequence-specific endonuclease cleavage site sequence, the first homology arm, the replacement DNA, the second homology arm, and a cleaved target sequence-specific endonuclease cleavage site sequence. In certain embodiments, the cleaved target sequence-specific endonuclease sequence can comprise a blunt DNA end or a blunt DNA end that can optionally comprise a 5′ phosphate group. In certain embodiments, the cleaved target sequence-specific endonuclease sequence comprises a DNA end having a single-stranded 5′ or 3′ DNA overhang. Such cleaved target sequence-specific endonuclease cleavage site sequences can be produced by either cleaving an intact target sequence-specific endonuclease cleavage site sequence or by synthesizing a copy of the cleaved target sequence-specific endonuclease cleavage site sequence. Donor DNA templates can be synthesized either chemically or enzymatically (e.g., in a polymerase chain reaction (PCR)).
Various treatments are useful in delivery of gene editing molecules and/or other molecules to a plant cell. In certain embodiments, one or more treatments is employed to deliver the gene editing or other molecules (e.g., comprising a polynucleotide, polypeptide or combination thereof) into a eukaryotic or plant cell, e.g., through barriers such as a cell wall, a plasma membrane, a nuclear envelope, and/or other lipid bilayer. In certain embodiments, a polynucleotide-, polypeptide-, or RNP-containing composition comprising the molecules are delivered directly, for example by direct contact of the composition with a plant cell. Aforementioned compositions can be provided in the form of a liquid, a solution, a suspension, an emulsion, a reverse emulsion, a colloid, a dispersion, a gel, liposomes, micelles, an injectable material, an aerosol, a solid, a powder, a particulate, a nanoparticle, or a combination thereof can be applied directly to a plant, plant part, plant cell, or plant explant (e.g., through abrasion or puncture or otherwise disruption of the cell wall or cell membrane, by spraying or dipping or soaking or otherwise directly contacting, by microinjection). For example, a plant cell or plant protoplast is soaked in a liquid genome editing molecule-containing composition, whereby the agent is delivered to the plant cell. In certain embodiments, the agent-containing composition is delivered using negative or positive pressure, for example, using vacuum infiltration or application of hydrodynamic or fluid pressure. In certain embodiments, the agent-containing composition is introduced into a plant cell or plant protoplast, e.g., by microinjection or by disruption or deformation of the cell wall or cell membrane, for example by physical treatments such as by application of negative or positive pressure, shear forces, or treatment with a chemical or physical delivery agent such as surfactants, liposomes, or nanoparticles; see, e.g., delivery of materials to cells employing microfluidic flow through a cell-deforming constriction as described in US Published Patent Application 2014/0287509, incorporated by reference in its entirety herein. Other techniques useful for delivering the agent-containing composition to a eukaryotic cell, plant cell or plant protoplast include: ultrasound or sonication; vibration, friction, shear stress, vortexing, cavitation; centrifugation or application of mechanical force; mechanical cell wall or cell membrane deformation or breakage; enzymatic cell wall or cell membrane breakage or permeabilization; abrasion or mechanical scarification (e.g., abrasion with carborundum or other particulate abrasive or scarification with a file or sandpaper) or chemical scarification (e.g., treatment with an acid or caustic agent); and electroporation. In certain embodiments, the agent-containing composition is provided by bacterially mediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.) transfection of the plant cell or plant protoplast with a polynucleotide encoding the genome editing molecules (e.g., RNA dependent DNA endonuclease, RNA dependent DNA binding protein, RNA dependent nickase, ABE, or CBE, and/or guide RNA); see, e.g., Broothaerts et al. (2005) Nature, 433:629-633). Any of these techniques or a combination thereof are alternatively employed on the plant explant, plant part or tissue or intact plant (or seed) from which a plant cell is optionally subsequently obtained or isolated; in certain embodiments, the agent-containing composition is delivered in a separate step after the plant cell has been isolated.
In some embodiments, one or more polynucleotides or vectors driving expression of one or more genome editing molecules or trait-conferring genes (e.g.; herbicide tolerance, insect resistance, and/or male sterility) are introduced into a plant cell. In certain embodiments, a polynucleotide vector comprises a regulatory element such as a promoter operably linked to one or more polynucleotides encoding genome editing molecules and/or trait-conferring genes. In such embodiments, expression of these polynucleotides can be controlled by selection of the appropriate promoter, particularly promoters functional in a eukaryotic cell (e.g., plant cell); useful promoters include constitutive, conditional, inducible, and temporally or spatially specific promoters (e.g., a tissue specific promoter, a developmentally regulated promoter, or a cell cycle regulated promoter). Developmentally regulated promoters that can be used in plant cells include Phospholipid Transfer Protein (PLTP), fructose-1,6-bisphosphatase protein, NAD(P)-binding Rossmann-Fold protein, adipocyte plasma membrane-associated protein-like protein, Rieske [2Fe-2S] iron-sulfur domain protein, chlororespiratory reduction 6 protein, D-glycerate 3-kinase, chloroplastic-like protein, chlorophyll a-b binding protein 7, chloroplastic-like protein, ultraviolet-B-repressible protein, Soul heme-binding family protein, Photosystem I reaction center subunit psi-N protein, and short-chain dehydrogenase/reductase protein that are disclosed in US Patent Application Publication No. 20170121722, which is incorporated herein by reference in its entirety and specifically with respect to such disclosure. In certain embodiments, the promoter is operably linked to nucleotide sequences encoding multiple guide RNAs, wherein the sequences encoding guide RNAs are separated by a cleavage site such as a nucleotide sequence encoding a microRNA recognition/cleavage site or a self-cleaving ribozyme (see, e.g., Ferré-D'Amaré and Scott (2014) Cold Spring Harbor Perspectives Biol., 2:a003574). In certain embodiments, the promoter is an RNA polymerase III promoter operably linked to a nucleotide sequence encoding one or more guide RNAs. In certain embodiments, the RNA polymerase III promoter is a plant U6 spliceosomal RNA promoter, which can be native to the genome of the plant cell or from a different species, e.g., a U6 promoter from maize, tomato, or soybean such as those disclosed U.S. Patent Application Publication 2017/0166912, or a homologue thereof; in an example, such a promoter is operably linked to DNA sequence encoding a first RNA molecule including a Cas12a gRNA followed by an operably linked and suitable 3′ element such as a U6 poly-T terminator. In another embodiment, the RNA polymerase III promoter is a plant U3, 7SL (signal recognition particle RNA), U2, or U5 promoter, or chimerics thereof, e.g., as described in U.S. Patent Application Publication 20170166912. In certain embodiments, the promoter operably linked to one or more polynucleotides is a constitutive promoter that drives gene expression in eukaryotic cells (e.g., plant cells). In certain embodiments, the promoter drives gene expression in the nucleus or in an organelle such as a chloroplast or mitochondrion. Examples of constitutive promoters for use in plants include a CaMV 35S promoter as disclosed in U.S. Pat. Nos. 5,858,742 and 5,322,938, a rice actin promoter as disclosed in U.S. Pat. No. 5,641,876, a maize chloroplast aldolase promoter as disclosed in U.S. Pat. No. 7,151,204, and the nopaline synthase (NOS) and octopine synthase (OCS) promoters from Agrobacterium tumefaciens. In certain embodiments, the promoter operably linked to one or more polynucleotides encoding elements of a genome-editing system is a promoter from figwort mosaic virus (FMV), a RUBISCO promoter, or a pyruvate phosphate dikinase (PPDK) promoter, which is active in photosynthetic tissues. Other contemplated promoters include cell-specific or tissue-specific or developmentally regulated promoters, for example, a promoter that limits the expression of the nucleic acid targeting system to germline or reproductive cells (e.g., promoters of genes encoding DNA ligases, recombinases, replicases, or other genes specifically expressed in germline or reproductive cells). In certain embodiments, the genome alteration is limited only to those cells from which DNA is inherited in subsequent generations, which is advantageous where it is desirable that expression of the genome-editing system be limited in order to avoid genotoxicity or other unwanted effects. All of the patent publications referenced in this paragraph are incorporated herein by reference in their entirety.
Expression vectors or polynucleotides provided herein may contain a DNA segment near the 3′ end of an expression cassette that acts as a signal to terminate transcription and directs polyadenylation of the resultant mRNA and may also support promoter activity. Such a 3′ element is commonly referred to as a “3′-untranslated region” or “3′-UTR” or a “polyadenylation signal.” In some cases, plant gene-based 3′ elements (or terminators) consist of both the 3′-UTR and downstream non-transcribed sequence (Nuccio et al., 2015). Useful 3′ elements include: Agrobacterium tumefaciens nos 3′, tml 3′, tmr 3′, tms 3′, ocs 3′, and tr7 3′ elements disclosed in U.S. Pat. No. 6,090,627, incorporated herein by reference, and 3′ elements from plant genes such as the heat shock protein 17, ubiquitin, and fructose-1,6-biphosphatase genes from wheat (Triticum aestivum), and the glutelin, lactate dehydrogenase, and beta-tubulin genes from rice (Oryza sativa), disclosed in US Patent Application Publication 2002/0192813 A1. All of the patent publications referenced in this paragraph are incorporated herein by reference in their entireties.
In certain embodiments, the plant cells can comprise haploid, diploid, or polyploid plant cells or plant protoplasts, for example, those obtained from a haploid, diploid, or polyploid plant, plant part or tissue, or callus. In certain embodiments, plant cells in culture (or the regenerated plant, progeny seed, and progeny plant) are haploid or can be induced to become haploid; techniques for making and using haploid plants and plant cells are known in the art, see, e.g., methods for generating haploids in Arabidopsis thaliana by crossing of a wild-type strain to a haploid-inducing strain that expresses altered forms of the centromere-specific histone CENH3, as described by Maruthachalam and Chan in “How to make haploid Arabidopsis thaliana”, protocol available at www[dot]openwetware[dot]org/images/d/d3/Haploid_Arabidopsis_protocol[dot]pdf; (Ravi et al. (2014) Nature Communications, 5:5334, doi: 10.1038/ncomms6334). Haploids can also be obtained in a wide variety of monocot plants (e.g., maize, wheat, rice, sorghum, barley) or dicot plants (e.g., soybean, Brassica sp. including canola, cotton, tomato) by crossing a plant comprising a mutated CENH3 gene with a wildtype diploid plant to generate haploid progeny as disclosed in U.S. Pat. No. 9,215,849, which is incorporated herein by reference in its entirety. Haploid-inducing maize lines that can be used to obtain haploid maize plants and/or cells include Stock 6, MHI (Moldovian Haploid Inducer), indeterminate gametophyte (ig) mutation, KEMS, RWK, ZEM, ZMS, KMS, and well as transgenic haploid inducer lines disclosed in U.S. Pat. No. 9,677,082, which is incorporated herein by reference in its entirety. Examples of haploid cells include but are not limited to plant cells obtained from haploid plants and plant cells obtained from reproductive tissues, e.g., from flowers, developing flowers or flower buds, ovaries, ovules, megaspores, anthers, pollen, megagametophyte, and microspores. In certain embodiments where the plant cell or plant protoplast is haploid, the genetic complement can be doubled by chromosome doubling (e.g., by spontaneous chromosomal doubling by meiotic non-reduction, or by using a chromosome doubling agent such as colchicine, oryzalin, trifluralin, pronamide, nitrous oxide gas, anti-microtubule herbicides, anti-microtubule agents, and mitotic inhibitors) in the plant cell or plant protoplast to produce a doubled haploid plant cell or plant protoplast wherein the complement of genes or alleles is homozygous; yet other embodiments include regeneration of a doubled haploid plant from the doubled haploid plant cell or plant protoplast. Another embodiment is related to a hybrid plant having at least one parent plant that is a doubled haploid plant provided by this approach. Production of doubled haploid plants provides homozygosity in one generation, instead of requiring several generations of self-crossing to obtain homozygous plants. The use of doubled haploids is advantageous in any situation where there is a desire to establish genetic purity (i.e. homozygosity) in the least possible time. Doubled haploid production can be particularly advantageous in slow-growing plants or for producing hybrid plants that are offspring of at least one doubled-haploid plant.
In certain embodiments, the plant cells used in the methods provided herein can include non-dividing cells. Such non-dividing cells can include plant cell protoplasts, plant cells subjected to one or more of a genetic and/or pharmaceutically induced cell-cycle blockage, and the like.
In certain embodiments, the plant cells in used in the methods provided herein can include dividing cells. Dividing cells can include those cells found in various plant tissues including leaves, meristems, and embryos. These tissues include dividing cells from young maize leaf, meristems and scutellar tissue from about 8 or 10 to about 12 or 14 days after pollination (DAP) embryos. The isolation of maize embryos has been described in several publications (Brettschneider, Becker, and Lorz 1997; Leduc et al. 1996; Frame et al. 2011; K. Wang and Frame 2009). In certain embodiments, basal leaf tissues (e.g., leaf tissues located about 0 to 3 cm from the ligule of a maize plant; Kirienko, Luo, and Sylvester 2012) are targeted for HDR-mediated gene editing. Methods for obtaining regenerable plant structures and regenerating plants from the NHEJ-, MMEJ-, or HDR-mediated gene editing of plant cells provided herein can be adapted from methods disclosed in US Patent Application Publication No. 20170121722, which is incorporated herein by reference in its entirety and specifically with respect to such disclosure. In certain embodiments, single plant cells subjected to the HDR-mediated gene editing will give rise to single regenerable plant structures. In certain embodiments, the single regenerable plant cell structure can form from a single cell on, or within, an explant that has been subjected to the NHEJ-, MMEJ-, or HDR-mediated gene editing.
In some embodiments, methods provided herein can include the additional step of growing or regenerating a plant from a plant cell that had been subjected to the improved HDR-mediated gene editing or from a regenerable plant structure obtained from that plant cell. In certain embodiments, the plant can further comprise an inserted transgene, a target gene edit, or genome edit as provided by the methods and compositions disclosed herein. In certain embodiments, callus is produced from the plant cell, and plantlets and plants produced from such callus. In other embodiments, whole seedlings or plants are grown directly from the plant cell without a callus stage. Thus, additional related aspects are directed to whole seedlings and plants grown or regenerated from the plant cell or plant protoplast having a target gene edit or genome edit, as well as the seeds of such plants. In certain embodiments wherein the plant cell or plant protoplast is subjected to genetic modification (for example, genome editing by means of, e.g., an RdDe), the grown or regenerated plant exhibits a phenotype associated with the genetic modification. In certain embodiments, the grown or regenerated plant includes in its genome two or more genetic or epigenetic modifications that in combination provide at least one phenotype of interest. In certain embodiments, a heterogeneous population of plant cells having a target gene edit or genome edit, at least some of which include at least one genetic or epigenetic modification, is provided by the method; related aspects include a plant having a phenotype of interest associated with the genetic or epigenetic modification, provided by either regeneration of a plant having the phenotype of interest from a plant cell or plant protoplast selected from the heterogeneous population of plant cells having a target gene or genome edit, or by selection of a plant having the phenotype of interest from a heterogeneous population of plants grown or regenerated from the population of plant cells having a targeted genetic edit or genome edit. Examples of phenotypes of interest include herbicide resistance, improved tolerance of abiotic stress (e.g., tolerance of temperature extremes, drought, or salt) or biotic stress (e.g., resistance to nematode, bacterial, or fungal pathogens), improved utilization of nutrients or water, modified lipid, carbohydrate, or protein composition, improved flavor or appearance, improved storage characteristics (e.g., resistance to bruising, browning, or softening), increased yield, altered morphology (e.g., floral architecture or color, plant height, branching, root structure). In an embodiment, a heterogeneous population of plant cells having a target gene edit or genome edit (or seedlings or plants grown or regenerated therefrom) is exposed to conditions permitting expression of the phenotype of interest; e.g., selection for herbicide resistance can include exposing the population of plant cells having a target gene edit or genome edit (or seedlings or plants grown or regenerated therefrom) to an amount of herbicide or other substance that inhibits growth or is toxic, allowing identification and selection of those resistant plant cells (or seedlings or plants) that survive treatment. Methods for obtaining regenerable plant structures and regenerating plants from plant cells or regenerable plant structures can be adapted from published procedures (Roest and Gilissen, Acta Bot. Neerl., 1989, 38(1), 1-23; Bhaskaran and Smith, Crop Sci. 30(6):1328-1337; Ikeuchi et al., Development, 2016, 143: 1442-1451). Methods for obtaining regenerable plant structures and regenerating plants from plant cells or regenerable plant structures can also be adapted from US Patent Application Publication No. 20170121722, which is incorporated herein by reference in its entirety and specifically with respect to such disclosure. Also provided are heterogeneous or homogeneous populations of such plants or parts thereof (e.g., seeds), succeeding generations or seeds of such plants grown or regenerated from the plant cells or plant protoplasts, having a target gene edit or genome edit. Additional related aspects include a hybrid plant provided by crossing a first plant grown or regenerated from a plant cell or plant protoplast having a target gene edit or genome edit and having at least one genetic or epigenetic modification, with a second plant, wherein the hybrid plant contains the genetic or epigenetic modification; also contemplated is seed produced by the hybrid plant. Also envisioned as related aspects are progeny seed and progeny plants, including hybrid seed and hybrid plants, having the regenerated plant as a parent or ancestor. The plant cells and derivative plants and seeds disclosed herein can be used for various purposes useful to the consumer or grower. In other embodiments, processed products are made from the plant or its seeds, including: (a) corn, soy, cotton, or canola seed meal (defatted or non-defatted); (b) extracted proteins, oils, sugars, and starches; (c) fermentation products; (d) animal feed or human food products (e.g., feed and food comprising corn, soy, cotton, or canola seed meal (defatted or non-defatted) and other ingredients (e.g., other cereal grains, other seed meal, other protein meal, other oil, other starch, other sugar, a binder, a preservative, a humectant, a vitamin, and/or mineral; (e) a pharmaceutical; (f) raw or processed biomass (e.g., cellulosic and/or lignocellulosic material); and (g) various industrial products.
Various embodiments of the plants, genomes, methods, biological samples, and other compositions described herein are set forth in the following sets of numbered embodiments.
1. An edited transgenic plant genome comprising a first modified transgenic locus that comprises:
(i) a first originator guide RNA recognition site (OgRRS) comprising a protospacer adjacent motif (PAM) site operably linked to a guide RNA hybridization site, wherein the OgRRS is located in transgenic DNA, in non-transgenic plant genomic DNA, or in a combination thereof in a first DNA junction polynucleotide of the first modified transgenic locus; and
(ii) a first cognate guide RNA recognition site (CgRRS) comprising a protospacer adjacent motif (PAM) site operably linked to a guide RNA hybridization site located in a second DNA junction polynucleotide of the first modified transgenic locus, wherein the CgRRS is absent from transgenic plant genomes comprising a first original transgenic locus that is unmodified and wherein the OgRRS and the CgRRS can hybridize to one first guide RNA (gRNA).
2. The edited transgenic plant genome of embodiment 1, wherein the guide RNA hybridization site of the OgRRS and the CgRRS comprise at least 18 nucleotides of identical DNA sequence.
3. The edited transgenic plant genome of embodiment 1, wherein the OgRRS and the CgRRS can be cleaved by the same RNA dependent DNA endonuclease (RdDe) when the OgRRS and the CgRRS are hybridized to the one first gRNA.
4. The edited transgenic plant genome of embodiment 3, wherein the RdDe is a class 2 type II or class 2 type V RdDe.
5. The edited transgenic plant genome of embodiment 1, wherein the PAM site in the OgRRS and the PAM site in the CgRRS comprise distinct PAM sequences that are recognized by the same RdDe when the OgRRS and the CgRRS are hybridized to the one gRNA.
6. The edited transgenic plant genome of any one of embodiments 1 to 5, wherein the OgRRS is located in non-transgenic DNA, in non-transgenic DNA and within about 1000, 750, 500, 250, 100, or 50 base pairs (bp) of transgenic DNA, or within transgenic DNA of the first DNA junction polynucleotide; and/or wherein the CgRRS is located within in non-transgenic DNA, in non-transgenic DNA and about 1000, 750, 500, 250, 100, or 50 bp of transgenic DNA in the second DNA junction polynucleotide, or within transgenic DNA of the second DNA junction polynucleotide.
7. The edited transgenic plant genome of any one of embodiments 1 to 6, wherein the edited transgenic plant genome further comprises a second modified transgenic locus that comprises:
(i) a second OgRRS located in transgenic DNA, in non-transgenic plant genomic DNA, or in a combination thereof in a first DNA junction polynucleotide of the second modified transgenic locus; and
(ii) a second CgRRS located in a second DNA junction polynucleotide of the second modified transgenic locus, wherein the CgRRS is absent from unedited transgenic plant genomes comprising a second original transgenic locus, optionally wherein the second OgRRS and the second CgRRS can hybridize to one second guide RNA (gRNA) and optionally wherein the second guide RNA is not complementary to the first OgRRS and the first CgRRS.
8. The edited transgenic plant genome of any one of embodiments 1 to 7, wherein the first and/or second modified transgenic locus comprises at least one modification of a 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, MON87403, MON87419, MON87460, MZHG0JG, MZIR098, VCO-Ø1981-5, 98140, or TC1507 original transgenic locus in a transgenic corn plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus and optionally the CgRRS in the second DNA junction polynucleotide of the second modified transgenic locus, and optionally wherein the modification further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus.
9. The edited transgenic plant genome of any one of embodiments 1 to 7, wherein the first and/or second modified transgenic locus comprises a modification of an A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, or SYHT0H2 original transgenic locus in a transgenic soybean plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus and optionally the CgRRS in the second DNA junction polynucleotide of the second modified transgenic locus, and wherein the modification optionally further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus.
10. The edited transgenic plant genome of any one of embodiments 1 to 7, wherein the first and/or second modified transgenic locus comprises at least one modification of a DAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701, or MON88913 original transgenic locus in a transgenic cotton plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus and optionally the CgRRS in the second DNA junction polynucleotide of the second modified transgenic locus, and wherein the modification optionally further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus.
11. The edited transgenic plant genome of any one of embodiments 1 to 7, wherein the first and/or second modified transgenic locus comprises a modification of an GT73, HCN28, MON88302, or MS8 original transgenic locus in a transgenic canola plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus and optionally the CgRRS in the second DNA junction polynucleotide of the second modified transgenic locus, and wherein the modification optionally further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus.
12. The edited transgenic plant genome of any one of embodiments to 1 to 11, wherein the first and/or second modified transgenic locus lacks a selectable marker transgene which confers resistance to an antibiotic, tolerance to an herbicide, or an ability to grow on a specific carbon source, optionally wherein the selectable marker transgene was present in the original transgenic locus and/or wherein the specific carbon source is optionally mannose.
13. The edited transgenic plant genome of any one of embodiments 1 to 12, wherein: (i) the first CgRRS is located in non-transgenic plant genomic DNA of the second DNA junction polynucleotide of the first modified transgenic locus; or (ii) the first CgRRS is located in transgenic DNA of the second DNA junction polynucleotide of the first modified transgenic locus.
14. The edited transgenic plant genome of embodiment 13, wherein the CgRRS is located in the non-transgenic plant genomic DNA and comprises a sequence having 50% to 70% sequence identity to the non-transgenic plant genomic DNA located at the same chromosomal location in the original transgenic locus.
15. The edited transgenic plant genome of any one of embodiments 1 to 14, wherein the first and/or second transgenic locus further comprise a second introduced transgene or optionally wherein the second introduced transgene is integrated at a site in the modified transgenic locus which was occupied by a selectable marker transgene in the original transgenic locus.
16. The edited transgenic plant genome of any one of embodiments 1 to 15, wherein the genome further comprises a targeted genetic change.
17. A transgenic plant cell comprising the edited transgenic plant genome of any one of embodiments 1 to 16.
18. A transgenic plant comprising the transgenic plant genome of any one of embodiments 1 to 16.
19. A transgenic plant part comprising the edited transgenic plant genome of any one of embodiments 1 to 16.
20. The transgenic plant part of embodiment 19, wherein the part is a seed, leaf, tuber, stem, root, or boll.
21. A method for obtaining a bulked population of inbred seed for commercial seed production comprising selfing the transgenic plant of embodiment 18 and harvesting seed from the selfed crop plants.
22. A method of obtaining hybrid crop seed comprising crossing a first crop plant comprising the transgenic plant of embodiment 18 to a second crop plant and harvesting seed from the cross.
23. The method of embodiment 22 wherein the first crop plant and the second crop plant are in distinct heterotic groups.
24. The method of embodiment 22 or 23, wherein either the first or second crop plant are pollen recipients which have been rendered male sterile.
25. The method of any one of embodiments 22 to 24, wherein the crop plant is rendered male sterile by emasculation, cytoplasmic male sterility, a chemical hybridizing agent or system, a transgene, and/or a mutation in an endogenous plant gene.
26. The method of any one of embodiments 22 to 25, further comprising the step of sowing the hybrid crop seed.
27. A DNA molecule comprising the cognate guide RNA recognition site (CgRRS) and at least 10 bp of transgenic DNA or non-transgenic plant genomic DNA flanking the CgRRS, wherein the transgenic DNA or non-transgenic plant genomic DNA comprises DNA sequences of the second DNA junction polynucleotide of the first modified transgenic locus set forth in any one of embodiments 1 to 11.
28. The DNA of embodiment 27, wherein the modified transgenic locus is a 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, MON87403, MON87419, MON87460, MZHG0JG, MZIR098, VCO-Ø1981-5, 98140, or TC1507 transgenic locus.
29. The DNA of embodiment 27, wherein the modified transgenic locus is an A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, and/or SYHT0H2 transgenic locus.
30. The DNA of embodiment 27 wherein the modified transgenic locus is: (i) a DAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701, and/or MON88913 transgenic locus and wherein the modifications optionally further comprise a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus; or (ii) wherein the modified transgenic locus is a GT73, HCN28, MON88302, or MS8 transgenic locus.
31. The DNA of any one of embodiments 27 to 30, wherein the DNA is purified or isolated.
32. A processed transgenic plant product containing the DNA of any one of embodiments 27 to 30.
33. A biological sample containing the DNA of any one of embodiments 27 to 30.
34. A nucleic acid marker adapted for detection of genomic DNA or fragments thereof comprising a cognate guide RNA recognition site (CgRRS) in, adjacent to, or operably linked to a DNA junction polynucleotide of a modified transgenic locus.
35. The nucleic acid marker of embodiment 34, comprising a polynucleotide of at least 18 nucleotides in length which spans DNA sequences comprising both the CgRRS and non-transgenic plant genomic DNA flanking either a telomere-proximal or a centromere proximal end of the CgRRS.
36. The nucleic acid marker of embodiment 34 or 35, wherein the marker further comprises a detectable label.
37. The nucleic acid marker of any one of embodiments 34 to 36, wherein the CgRRS and non-transgenic plant genomic DNA are identical to sequences in a modified transgenic locus.
38. The nucleic acid marker of any one of embodiments 34 to 37, wherein the modified transgenic locus is a 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, MON87403, MON87419, MON87460, MZHG0JG, MZIR098, VCO-Ø1981-5, 98140, or TC1507 transgenic locus comprising the CgRRS, wherein the CgRRS is located in, adjacent to, or operably linked to a DNA junction polynucleotide of the modified transgenic locus.
39. The nucleic acid marker of any one of embodiments 34 to 37, wherein the modified transgenic locus is a modified A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, or SYHT0H2 transgenic locus comprising the CgRRS, wherein the CgRRS is located in, adjacent to, or operably linked to a DNA junction polynucleotide of the modified transgenic locus.
40. The nucleic acid marker of any one of embodiments 34 to 37, wherein the modified transgenic locus is a DAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701, and/or MON88913 transgenic locus comprising the CgRRS, wherein the CgRRS is located in, adjacent to, or operably linked to a DNA junction polynucleotide of the modified transgenic locus.
41. The nucleic acid marker of any one of embodiments 34 to 37, wherein the modified transgenic locus is a GT73, HCN28, MON88302, or MS8 transgenic locus comprising the CgRRS, wherein the CgRRS is located in, adjacent to, or operably linked to a DNA junction polynucleotide of the modified transgenic locus.
42. A processed transgenic plant product obtained from the transgenic plant part of embodiment 19 or 20, wherein the processed plant product contains a polynucleotide comprising a cognate guide RNA recognition site (CgRRS) and at least 10 bp of transgenic DNA or non-transgenic plant genomic DNA flanking the CgRRS, wherein the transgenic DNA or non-transgenic plant genomic DNA comprises transgenic or non-transgenic plant genomic DNA sequences of the second DNA junction polynucleotide of the first modified transgenic locus.
43. A biological sample obtained from the transgenic plant cell of embodiment 17, the transgenic plant of embodiment 18, or the transgenic plant part of embodiment 19 or 20, wherein the biological sample contains one or more polynucleotide(s) comprising the a cognate guide RNA recognition site (CgRRS) and at least 10 bp of transgenic DNA or non-transgenic plant genomic DNA flanking the CgRRS, wherein the transgenic DNA or non-transgenic plant genomic DNA comprises transgenic or non-transgenic plant genomic DNA sequences of the second DNA junction polynucleotide of the first modified transgenic locus.
44. A method of detecting the edited transgenic plant genome of any one of embodiments 1 to 16, comprising the step of detecting the presence of a polynucleotide comprising one or more of said CgRRS.
45. The method of embodiment 44, wherein the polynucleotide is detected by detecting a single nucleotide polymorphism (SNP) in the CgRRS that is present in the modified transgenic locus but absent in the original transgenic locus.
46. The method of embodiment 44 or 45, wherein the edited transgenic plant genome is detected in a transgenic plant cell, a transgenic plant part, a transgenic plant, a processed transgenic plant product, or a biological sample.
47. A method of obtaining an edited transgenic plant genome comprising a modified transgenic locus comprising the step of introducing a cognate guide RNA recognition site (CgRRS) in a DNA junction polynucleotide of an original transgenic locus, wherein the CgRRS is in, adjacent to, or operably linked to a DNA junction polynucleotide of the modified transgenic locus.
48. The method of embodiment 47, wherein the CgRRS is located in non-transgenic plant genomic DNA of the DNA junction polynucleotide of the modified transgenic locus and optionally wherein the CgRRS comprises a sequence having 50% to 70% sequence identity to the non-transgenic plant genomic DNA located at the same chromosomal location in the original transgenic locus.
49. The method of embodiment 47 or 48, wherein the CgRRS is introduced by:
(a) contacting the original transgenic locus with: (i) an RNA dependent DNA endonuclease (RdDe) or RdDe nickase; and a guide RNA comprising an RNA equivalent of the DNA located immediately 5′ or 3′ to an original PAM site located within the DNA junction polynucleotide of the original transgenic locus; or (ii) a guide RNA comprising an RNA equivalent of the DNA located immediately 5′ or 3′ to an original PAM site located within the DNA junction polynucleotide of the original transgenic locus; and (iii) a donor DNA template spanning a double stranded DNA break site in the DNA junction polynucleotide, wherein the donor DNA template comprises a guide RNA hybridization site of the CgRRS, optionally a PAM site of the CgRRS, and optionally DNA sequences of the DNA junction polynucleotide flanking the double stranded DNA break site; and
(b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant comprising the CgRRS.
50. The method of embodiment 47 or 48, wherein the CgRRS is introduced by:
(a) contacting the original transgenic locus with: (i) at least one adenine base editor (ABE) and/or cytosine base pair editor (CBE); and (ii) a guide RNA comprising an RNA equivalent of the DNA located immediately 5′ or 3′ to an original PAM site located within the DNA junction polynucleotide of the original transgenic locus; and
(b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant comprising the CgRRS.
51. The method of embodiment 47 or 48, wherein the CgRRS is introduced by:
(a) contacting the original transgenic locus with: (i) a Zinc Finger Nuclease or TALEN which recognizes a DNA junction polynucleotide of the original transgenic locus or (ii) a Zinc Finger nickase or Tale nickase which recognizes a DNA junction polynucleotide of the original transgenic locus; and (iii) a donor DNA template spanning a double stranded DNA break site in the DNA junction polynucleotide, wherein the donor DNA template comprises a guide RNA hybridization site of the CgRRS, optionally a PAM site of the CgRRS, and optionally DNA sequences of the DNA junction polynucleotide flanking the double stranded DNA break site; and
(b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant comprising the CgRRS.
52. The method of any one of embodiments 47 to 51, further comprising contacting the original transgenic locus with one or more gene editing molecules that provide for excision or inactivation of a selectable marker transgene of the original transgenic locus and selecting for a transgenic plant cell, transgenic plant part, or transgenic plant wherein the selectable marker transgene has been excised or inactivated.
53. The method of embodiment 52, wherein the gene editing molecules include a donor DNA template or other DNA template containing an expression cassette or coding region which confers a useful trait and the transgenic plant cell, transgenic plant part, or transgenic plant is selected for integration of the expression cassette at the site of the selectable marker transgene excision or inactivation.
54. A method of excising a modified transgenic locus or portion thereof from an edited transgenic plant genome comprising the steps of:
(a) contacting the edited transgenic plant genome of any one of embodiments 1 to 11 with: (i) an RNA dependent DNA endonuclease (RdDe); and (ii) a guide RNA (gRNA) capable of hybridizing to the guide RNA hybridization site of the first OgRRS and the first CgRRS; wherein the RdDe recognizes a OgRRS/gRNA and a CgRRS/gRNA hybridization complex; and,
(b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant wherein the modified transgenic locus or a portion thereof flanked by the first OgRRS and the first CgRRS has been excised.
55. The method of embodiment 54, wherein the edited transgenic plant genome is contacted in step (a) by introducing one or more compositions comprising or encoding the RdDe(s) and the gRNA into a transgenic plant cell comprising the edited transgenic plant genome.
56. The method of embodiment 54 or 55, wherein the edited transgenic plant genome is contacted in step (a) by introducing one or more compositions comprising the gRNA into a transgenic plant cell comprising the edited transgenic plant genome, wherein the transgenic plant cell comprises a polynucleotide that encodes the RdDe and the encoded RdDe is expressed in the transgenic plant cell.
57. The method of any one of embodiments 54 to 56, wherein the edited transgenic plant genome further comprises a second modified transgenic locus that comprises a second OgRRS located in a first DNA junction polynucleotide of the second modified transgenic locus; and a second CgRRS located in a second DNA junction polynucleotide of the second modified transgenic locus; and wherein the method further comprises:
(a) contacting the edited transgenic plant genome with: (i) an RNA dependent DNA endonuclease (RdDe); and (ii) a guide RNA (gRNA) capable of hybridizing to the guide RNA hybridization site of the second OgRRS and the second CgRRS; wherein the RdDe recognizes a second OgRRS/gRNA and a second CgRRS/gRNA hybridization complex; and,
(b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant wherein the second modified transgenic locus or a portion thereof flanked by the second OgRRS and the second CgRRS has been excised.
58. The method of any one of embodiments 54 to 57, wherein the transgenic plant cell is in tissue culture, in a callus culture, a plant part, or in a whole plant and/or wherein the transgenic plant cell is a haploid plant cell.
59a. The method of any one of embodiments 54 to 58, wherein frequency of excision of the modified transgenic locus or a portion thereof in the transgenic plant cells, transgenic plant parts, or transgenic plants subjected to selection is greater than the frequency of excision of the modified transgenic locus or a portion thereof in control transgenic plant cells, transgenic plant parts, or transgenic plants treated with two control gRNAs and subjected to selection.
59b. The method of any one of embodiments 54 to 58, wherein the edited transgenic plant genome is further contacted in step (a) with a donor DNA template, optionally wherein the donor DNA template comprises a polynucleotide sequence comprising non-transgenic plant genomic DNA located outside of the modified transgenic DNA which is to be excised, optionally wherein the donor DNA template comprises non-transgenic plant genomic DNA present in an unmodified plant at the site where the transgenic locus was originally inserted, and optionally in step (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant wherein polynucleotides in any of the donor DNA templates have integrated in the position in the plant genome where the modified transgenic locus was excised.
60. A method of obtaining a plant breeding line comprising:
(a) crossing two transgenic plants comprising the edited transgenic genomes of any of embodiments 1 to 11, wherein a first plant comprising the first modified transgenic locus is crossed to a second plant comprising the second modified transgenic locus; and,
(b) selecting a progeny plant comprising the first and second modified transgenic locus from the cross, thereby obtaining a plant breeding line.
61. The method of embodiment 60, wherein the plant breeding line is subjected to a haploid inducer and a haploid plant breeding line comprising at least the first and second breeding line is selected.
62. A method for obtaining inbred transgenic plant germplasm containing different transgenic traits comprising:
(a) introgressing at least a first transgenic locus and a second transgenic locus into inbred germplasm to obtain a donor inbred parent plant line comprising the first and second transgenic loci, wherein a first OgRRS and a first CgRRS are operably linked to both DNA junction polynucleotides of at least the first transgenic locus and optionally wherein a second OgRRS and a second CgRRS are operably linked to the second transgenic locus;
(b) contacting the transgenic plant genome of the donor inbred parent plant line with: (i) an RNA dependent DNA endonuclease (RdDe); and (ii) a guide RNA (gRNA) capable of hybridizing to the guide RNA hybridization site of the first OgRRS and the second CgRRS; wherein the RdDe recognizes a first OgRRS/gRNA and a first CgRRS/gRNA hybridization complex; and
(c) selecting a transgenic plant cell, transgenic plant part, or transgenic plant comprising an edited transgenic plant genome in the inbred germplasm, wherein the first transgenic locus has been excised and the second transgenic locus is present in the inbred germplasm.
63. The method of embodiment 62, wherein the introgression comprises crossing germplasm comprising the first and/or second transgenic plant locus with the inbred germplasm, selecting progeny comprising the first or second transgenic plant locus, and crossing the selected progeny with the inbred germplasm as a recurrent parent.
64. The method of embodiment 62 or 63, further comprising contacting the transgenic plant genome in step (b) with one or more gene editing molecules that provide for excision or inactivation of a selectable marker transgene of the second transgenic locus and selecting for a transgenic plant cell, transgenic plant part, or transgenic plant wherein the selectable marker transgene has been excised or inactivated.
65. The method of any one of embodiments 62 to 64, wherein the gene editing molecules include a donor DNA template containing an expression cassette or coding region which confers a useful trait and the transgenic plant cell, transgenic plant part, or transgenic plant is selected for integration of the expression cassette at the site of the selectable marker transgene excision or inactivation.
66. The method of any one of embodiments 62 to 65, wherein a second OgRRS and a second CgRRS are operably linked to the second transgenic locus and wherein the method further comprises contacting the transgenic plant genome of the donor inbred parent plant line with: (i) an RNA dependent DNA endonuclease (RdDe); and (ii) a guide RNA (gRNA) capable of hybridizing to the guide RNA hybridization site of the second OgRRS and the second CgRRS; wherein the RdDe recognizes a second OgRRS/gRNA and a second CgRRS/gRNA hybridization complex in step (b); and selecting a transgenic plant cell, transgenic plant part, or transgenic plant wherein the second transgenic locus has been excised in step (c).
67. The method of any one of embodiments 62 to 66, wherein the transgenic plant genome is contacted in step (b) by introducing one or more compositions comprising or encoding the RdDe(s) and gRNAs into a transgenic plant cell comprising the transgenic plant genome.
68. The method of any one of embodiments 62 to 66, wherein the edited transgenic plant genome is contacted in step (a) by introducing one or more compositions comprising the gRNA into a transgenic plant cell comprising the edited transgenic plant genome, wherein the transgenic plant cell comprises a polynucleotide that encodes the RdDe and the encoded RdDe is expressed in the transgenic plant cell.
69. The method of any one of embodiments 62 to 68, wherein the transgenic plant genome is further contacted in step (b) with a donor DNA template molecule comprising an introduced transgene and a transgenic plant cell comprising an edited transgenic plant genome comprising an insertion of the introduced transgene in the first transgenic locus is selected in step (c).
70. The method of any one of embodiments 62 to 69, wherein the transgenic plant genome is further contacted in step (b) with: (i) a donor DNA template molecule comprising an introduced transgene; and (ii) one or more DNA editing molecules which introduce a double stranded DNA break in the second transgenic locus; and a transgenic plant cell comprising an edited transgenic plant genome comprising an insertion of the introduced transgene in the second transgenic locus is selected in step (b).
71. The method of any one of embodiments 62 to 70, further comprising:
(d) contacting the edited transgenic plant genome in the selected transgenic plant cell of step (c) with: (i) a donor DNA template molecule comprising an introduced transgene; and (ii) one or more DNA editing molecules which introduce a double stranded DNA break in or near the excision site of the first transgenic locus or in the second transgenic locus; and,
(e) selecting a transgenic plant cell, transgenic plant part, or transgenic plant comprising a further edited transgenic plant genome comprising an insertion of the introduced transgene in or near the excision site of the first transgenic locus or in the second transgenic locus.
72. The method of any one of embodiments 62 to 71, wherein the transgenic plant germplasm is transgenic corn plant germplasm and wherein the first and/or second modified transgenic locus comprises at least one modification of a 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, MON87403, MON87419, MON87460, MZHG0JG, MZIR098, VCO-Ø1981-5, 98140, or TC1507 original transgenic locus in a transgenic corn plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus and optionally the CgRRS in the second DNA junction polynucleotide of the transgenic locus, and wherein the modification optionally further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus.
73. The method of any one of embodiments 62 to 72, wherein the transgenic plant germplasm is transgenic soybean plant germplasm and wherein the first and/or second modified transgenic locus comprises a modification of an A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, or SYHT0H2 original transgenic locus in a transgenic soybean plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus and optionally the CgRRS in the second DNA junction polynucleotide of the transgenic locus, and wherein the modification optionally further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus.
74. The method of any one of embodiments 62 to 72, wherein the transgenic plant germplasm is transgenic cotton plant germplasm and wherein the first, second, and/or third modified transgenic locus comprises at least one modification of a DAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701, or MON88913 original transgenic locus in a transgenic cotton plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus and optionally the CgRRS in the second DNA junction polynucleotide of the transgenic locus, and wherein the modification optionally further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus.
75. The method of any one of embodiments 62 to 72, wherein the transgenic plant germplasm is transgenic canola plant germplasm and wherein the first, second, and or third modified transgenic locus comprises a modification of an GT73, HCN28, MON88302, or MS8 original transgenic locus in a transgenic canola plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus and optionally the CgRRS in the second DNA junction polynucleotide of the transgenic locus, and wherein the modification optionally further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus.
Transgenic plant genomes containing one or more of the following transgenic loci (events) are contacted with:
(i) an ABE or CBE and guide RNAs which recognize the indicated target DNA sites (guide RNA coding plus PAM site) in the 5′ or 3′ junction polynucleotides of the event to introduce a CgRRS in the junction polynucleotide; or
(ii) an RdDe and guide RNAs which recognize the indicated target DNA site (guide RNA coding plus PAM site) in the 5′ or 3′ junction polynucleotides of the event as well as a donor DNA template spanning the double stranded DNA break site in the junction polynucleotide to introduce a CgRRS in a junction polynucleotide. Plant cells, callus, parts, or whole plants comprising the introduced CgRRS in the transgenic plant genome are selected.
Two plant gene expression vectors are prepared. Plant expression cassettes for expressing a bacteriophage lambda exonuclease, a bacteriophage lambda beta SSAP protein, and an E. coli SSB are constructed essentially as set forth in US Patent Application Publication 20200407754, which is incorporated herein by reference in its entirety. A DNA sequence encoding a tobacco c2 nuclear localization signal (NLS) is fused in-frame to the DNA sequences encoding the exonuclease, the bacteriophage lambda beta SSAP protein, and the E. coli SSB to provide a DNA sequence encoding the c2 NLS-Exo, c2 NLS lambda beta SSAP, and c2 NLS-SSB fusion proteins that are set forth in SEQ ID NO: 135, SEQ ID NO: 134, and SEQ ID NO: 133 of US Patent Application Publication 20200407754, respectively, and incorporated herein by example. DNA sequences encoding the c2 NLS-Exo, c2 NLS lambda beta SSAP, and c2NLS-SSB fusion proteins are operably linked to a OsUBI1, ZmUBI1, OsACT promoter and a OsUbi1, ZmUBI1, OsACT polyadenylation site respectively, to provide the exonuclease, SSAP, and SSB plant expression cassettes.
A donor DNA template sequence (SEQ ID NO: 37) that targets the 5′ DNA junction polynucleotide of the MIR162 event for insertion of a 27 base pair heterologous sequence, that is identical to a Cas12a recognition site at the 3′-junction of the MIR162 T-DNA insert, by HDR is constructed. The donor DNA template sequence includes a replacement template with desired insertion region (27 base pair long sequence of SEQ ID NO: 35) flanked on both sides by homology arms of about 500-635 bp in length. The homology arms match (i.e., are homologous to) gDNA (genomic DNA) regions flanking the target gDNA insertion site. The replacement template region comprising the donor DNA template is flanked at each end by DNA sequences identical to the MIR162 5′ polynucleotide sequence recognized by an RNA-guided nuclease and one or more gRNA(s) (e.g. gRNAs encoded by SEQ ID NO: 38, 39, and 40). In certain cases, a double-stranded is made in the targeted MIR162 5′ polynucleotide sequence (e.g., using a Cas12a endonuclease and a gRNA encoded by SEQ ID NO: 38, 39, or 40) to allow for HDR with the donor DNA template and introduction of the CgRRS. In certain cases, a deletion is made in the targeted MIR162 5′ polynucleotide sequence (e.g., using a Cas12a endonuclease and gRNAs encoded by SEQ ID NO: 38 and 39 in combination or by using gRNAs encoded by SEQ ID NO: 39 and 40 in combination) to allow for HDR with the donor DNA template and introduction of the CgRRS.
A plant expression cassette that provides for expression of the RNA-guided sequence-specific Cas12a endonuclease is constructed. A plant expression cassette that provides for expression of a guide RNA complementary to sequences adjacent to the insertion site (e.g. gRNAs encoded by SEQ ID NO: 38, 39, and 40) are constructed. An Agrobacterium superbinary plasmid transformation vector containing a cassette that provides for the expression of the phosphinothricin N-acetyltransferase synthase (PAT) protein is constructed. Once the cassettes, donor sequence and Agrobacterium superbinary plasmid transformation vector are constructed, they were combined to generate two maize transformation plasmids.
A maize transformation plasmid is constructed with the PAT cassette, the RNA-guided sequence-specific endonuclease cassette, the guide RNA cassette, and the MIR162 5′-junction polynucleotide donor DNA template sequence into the Agrobacterium superbinary plasmid transformation vector (the control vector).
A maize transformation plasmid is constructed with the PAT cassette, the RNA-guided sequence-specific endonuclease cassette, the guide RNA cassette, the SSB cassette, the lambda beta SSAP cassette, the Exo cassette, and the MIR162 5′-junction polynucleotide donor DNA template into the Agrobacterium superbinary plasmid transformation vector (the lambda red vector).
All constructs are transformed into Agrobacterium strain LBA4404.
Maize transformations are performed based on published methods (Ishida et. al, Nature Protocols 2007; 2, 1614-1621). Briefly, immature embryos from inbred line GIBE0104, approximately 1.8-2.2 mm in size, are isolated from surface sterilized ears 10-14 days after pollination. Embryos are placed in an Agrobacterium suspension made with infection medium at a concentration of OD 600=1.0. Acetosyringone (200 μM) is added to the infection medium at the time of use. Embryos and Agrobacterium are placed on a rocker shaker at slow speed for 15 minutes. Embryos are then poured onto the surface of a plate of co-culture medium. Excess liquid media is removed by tilting the plate and drawing off all liquid with a pipette. Embryos are flipped as necessary to maintain a scutelum up orientation. Co-culture plates are placed in a box with a lid and cultured in the dark at 22° C. for 3 days. Embryos are then transferred to resting medium, maintaining the scutellum up orientation. Embryos remain on resting medium for 7 days at 27-28° C. Embryos that produced callus are transferred to Selection 1 medium with 7.5 mg/L phosphinothricin (PPT) and cultured for an additional 7 days. Callused embryos are placed on Selection 2 medium with 10 mg/L PPT and cultured for 14 days at 27-28° C. Growing calli resistant to the selection agent are transferred to Pre-Regeneration media with 10 mg/L PPT to initiate shoot development. Calli remained on Pre-Regeneration media for 7 days. Calli beginning to initiate shoots are transferred to Regeneration medium with 7.5 mg/L PPT in Phytatrays and cultured in light at 27-28° C. Shoots that reached the top of the Phytatray with intact roots are isolated into Shoot Elongation medium prior to transplant into soil and gradual acclimatization to greenhouse conditions.
When a sufficient amount of viable tissue is obtained, it can be screened for insertion at the MIR162 junction sequence, using a PCR-based approach. The PCR primer on the 5′-end can be 5′-ttttatgtattatttggtccctaca-3′ (SEQ ID NO: 41) and the. PCR primer on the 3′-end is 5′-gtcgacggcgtttaacaggctggca-3′ (SEQ ID NO: 42). These primers that flank donor DNA homology arms are used to amplify the MIR162 5′-junction sequence. The correct donor sequence insertion will produce a 1579 bp product. Amplicons can be sequenced directly using an amplicon sequencing approach or ligated to a convenient plasmid vector for Sanger sequencing. The CgRRS which is introduced into a 5′ junction polynucleotide of MIR162 comprises SEQ ID NO: 43. Those plants in which the MIR162 5′ junction polynucleotide sequence now contains the intended CgRRS (i.e. Cas12a recognition sequence) are selected and grown to maturity. The T-DNA encoding the Cas12a reagents can be segregated away from the modified junction sequence in a subsequent generation. The resultant INIR12 transgenic locus comprising the CgRRS and OgRRS (e.g. which each comprise SEQ ID NO: 35 and an operably linked PAM site) can be excised using Cas12a and a suitable gRNA which hybridizes to DNA comprising SEQ ID NO: 35 at both the OgRRS and the CgRRS.
Two plant gene expression vectors are prepared. Plant expression cassettes for expressing a bacteriophage lambda exonuclease, a bacteriophage lambda beta SSAP protein, and an E. coli SSB are constructed essentially as set forth in US Patent Application Publication 20200407754, which is incorporated herein by reference in its entirety and as described in Example 2.
A DNA donor sequence (SEQ ID NO: 47) that targets the 3′-T-DNA junction polynucleotide of the DP-4114 event (SEQ ID NO:1;
A plant expression cassette that provides for expression of the RNA-guided sequence-specific Cas12a endonuclease is constructed. A plant expression cassette that provides for expression of a guide RNA (e.g., encoded by SEQ ID NO: 49) complementary to sequences adjacent to the insertion site is constructed. An Agrobacterium superbinary plasmid transformation vector containing a cassette that provides for the expression of the phosphinothricin N-acetyltransferasesynthase (PAT) protein is constructed. Once the cassettes, donor sequence and Agrobacterium superbinary plasmid transformation vector are constructed, they are combined to generate two maize transformation plasmids.
A maize transformation plasmid is constructed with the PAT cassette, the RNA-guided sequence-specific endonuclease cassette, the guide RNA cassette, and the DP-4114 3′-T_DNA junction sequence DNA donor sequence into the Agrobacterium superbinary plasmid transformation vector (the control vector).
A maize transformation plasmid is constructed with the PAT cassette, the RNA-guided sequence-specific endonuclease cassette, the guide RNA cassette, the SSB cassette, the lambda beta SSAP cassette, the Exo cassette, and the DP-4114 3′-T_DNA junction sequence DNA donor sequence (SEQ ID NO: 47) into the Agrobacterium superbinary plasmid transformation vector (the lambda red vector).
All constructs are transformed into Agrobacterium strain LBA4404.
Maize transformations are performed based on published methods (Ishida et. al, Nature Protocols 2007; 2, 1614-1621). Briefly, immature embryos from inbred line GIBE0104, approximately 1.8-2.2 mm in size, are isolated from surface sterilized ears 10-14 days after pollination. Embryos are placed in an Agrobacterium suspension made with infection medium at a concentration of OD 600=1.0. Acetosyringone (200 μM) is added to the infection medium at the time of use. Embryos and Agrobacterium are placed on a rocker shaker at slow speed for 15 minutes. Embryos are then poured onto the surface of a plate of co-culture medium. Excess liquid media is removed by tilting the plate and drawing off all liquid with a pipette. Embryos are flipped as necessary to maintain a scutelum up orientation. Co-culture plates are placed in a box with a lid and cultured in the dark at 22° C. for 3 days. Embryos are then transferred to resting medium, maintaining the scutellum up orientation. Embryos remain on resting medium for 7 days at 27-28° C. Embryos that produced callus are transferred to Selection 1 medium with 7.5 mg/L phosphinothricin (PPT) and cultured for an additional 7 days. Callused embryos are placed on Selection 2 medium with 10 mg/L PPT and cultured for 14 days at 27-28° C. Growing calli resistant to the selection agent are transferred to Pre-Regeneration media with 10 mg/L PPT to initiate shoot development. Calli remained on Pre-Regeneration media for 7 days. Calli beginning to initiate shoots are transferred to Regeneration medium with 7.5 mg/L PPT in Phytatrays and cultured in light at 27-28° C. Shoots that reached the top of the Phytatray with intact roots are isolated into Shoot Elongation medium prior to transplant into soil and gradual acclimatization to greenhouse conditions.
When a sufficient amount of viable tissue is obtained, it can be screened for insertion at the DP-4114 junction sequence, using a PCR-based approach. The PCR primer on the 5′-end is 5′-tacgctgggccctggaaggctagga-3′ (SEQ ID NO: 50). The PCR primer on the 3′-end is 5′-gatggacgagacgaggcggtggaga-3′ (SEQ ID NO: 51). The above primers that flank donor DNA homology arms are used to amplify the DP-4114 3′-junction polynucleotide sequence. The correct donor sequence insertion will produce a 1563 bp product. A unique DNA fragment comprising the CgRRS in the DP-4114 3′ junction polynucleotide is set forth in SEQ ID NO: 52. Amplicons can be sequenced directly using an amplicon sequencing approach or ligated to a convenient plasmid vector for Sanger sequencing. Those plants in which the DP-4114 junction sequence now contains the intended Cas12a recognition sequence are selected and grown to maturity. The T-DNA encoding the Cas12a reagents can be segregated away from the modified junction sequence in a subsequent generation. The resultant INIR6 transgenic locus (SEQ ID NO: 53) comprising the CgRRS and OgRRS (e.g. which each comprise SEQ ID NO: 48) can be excised using Cas12a and a suitable gRNA which hybridizes to DNA comprising SEQ ID NO: 48 at both the OgRRS and the CgRRS.
The breadth and scope of the present disclosure should not be limited by any of the above-described embodiments.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/043207 | 7/26/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63059813 | Jul 2020 | US | |
| 63059860 | Jul 2020 | US | |
| 63059916 | Jul 2020 | US | |
| 63059963 | Jul 2020 | US | |
| 63199930 | Feb 2021 | US | |
| 63199949 | Feb 2021 | US | |
| 63199951 | Feb 2021 | US | |
| 63201029 | Apr 2021 | US | |
| 63201030 | Apr 2021 | US | |
| 63202569 | Jun 2021 | US | |
| 63203137 | Jul 2021 | US |
