TAL effector nucleases for gene editing

Abstract

Disclosed are TALEN compositions and methods of use, including using multiplexed compositions to simultaneously create targeted mutations in several genes, such as FAD3A / B / C genes, compositions for creating targeted mutations in a single gene, such as the gene encoding FAD2 protein, and combinations thereof.The compositions and methods can provide gene-edited plants, plant parts, and plant cells with improved characteristics compared to corresponding unaltered plants, plant parts, or plant cells.For example, soybean plants, plant parts, and plant cells are also provided that can produce seeds containing oil with relatively higher oleic acid levels and lower linoleic and linolenic acid levels than corresponding seeds that lack targeted mutations.

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on November 19, 2020, is named 1702_029PCT1_SL.txt and is 47,037 bytes in size. [Background technology]

[0002] Gene editing with infrequently cutting endonucleases can be used to generate deletions, insertions, and initiate homologous recombination. Transcription activator-like effector nucleases (TALENs) are infrequently cutting endonucleases that can target specific sequences and generate precise cuts in DNA. One TALEN design process assumes a left half TALEN (HT) that recognizes approximately 15 base pairs (bp), followed by a spacer region of approximately 15-18 bp, and a right HT recognition sequence of approximately 15 bp (see, e.g., U.S. Patent No. 8,450,471). This approach provides a high level of specificity for the intended target; however, multiple gene editing capabilities would be advantageous. Summary of the Invention [Problem to be solved by the invention]

[0003] Aspects of the present disclosure relate to compositions and methods for editing genes (e.g., by introducing mutations) using infrequently-cutting endonucleases such as TALENs.

[0004] In one aspect, provided herein is a composition comprising a first nucleic acid encoding a first transcription activator-like (TAL) effector nuclease monomer capable of binding to a first half-site sequence of a first target gene, and a set of second nucleic acids. Each second nucleic acid encodes a second transcription activator-like (TAL) effector nuclease monomer capable of binding to a second half-site sequence of the first target gene or a set of second target genes. In this composition, the first half-site sequence is a conserved sequence, and the first half-site sequence and each second half-site sequence are different and separated by a spacer sequence, and the first TAL effector nuclease monomer can form a dimer with each of the second TAL effector nucleases, thus providing a set of TALENs.

[0005] In some embodiments, when a first TAL effector nuclease monomer binds to a first half-site sequence and a second TAL effector nuclease monomer binds to a second half-site sequence, the dimer can cleave a target gene in a living cell.

[0006] In some embodiments, the first half-site sequence is a 100% conserved sequence. In some embodiments, the spacer sequence is about 15 to about 18 nucleotides in length.

[0007] In some embodiments, the first nucleic acid comprises a FokI endonuclease domain. In some embodiments, each second nucleic acid comprises a FokI endonuclease domain.

[0008] In some embodiments, the first nucleic acid is in a vector. In some embodiments, each second nucleic acid is in a vector. In some embodiments, the first nucleic acid and the set of second nucleic acids encoding the second TAL effector nuclease monomers are in a single vector.

[0009] In some embodiments, the first nucleic acid is mRNA in a plasmid. In some embodiments, each second nucleic acid is mRNA in a plasmid. In some embodiments, the set of the first nucleic acid and the second nucleic acid is mRNA in a plasmid.

[0010] In some embodiments, the set of second nucleic acids comprises three or more second nucleic acids. In some embodiments, the set of second nucleic acids comprises four or more second nucleic acids.

[0011] In some embodiments, the first target gene is a gene of the FAD3 gene family in Glycine max. In some embodiments, each second target gene is a gene of the FAD3 gene family in Glycine max.

[0012] In some embodiments, the first target gene is an allele of the Glycine max FAD3A gene. In some embodiments, the first target gene is an allele of the Glycine max FAD3B gene. In some embodiments, the first target gene is an allele of the Glycine max FAD3C gene. In some embodiments, the second target gene is an allele of the Glycine max FAD3A gene. In some embodiments, the second target gene is an allele of the Glycine max FAD3B gene. In some embodiments, the second target gene is an allele of the Glycine max FAD3C gene.

[0013] In some embodiments, the first half-site sequence is SEQ ID NO:18. In some embodiments, the composition herein comprises two second nucleic acids, each encoding a second transcription activator-like (TAL) effector nuclease monomer capable of binding to a second half-site sequence, wherein the second half-site sequence is SEQ ID NO: 17 or SEQ ID NO: 19.

[0014] In some embodiments, the composition further comprises one or more rare-cutting endonucleases targeting alleles of the FAD2-1 gene family of Glycine max. The one or more rare-cutting endonucleases can be TAL effector nucleases targeting FAD2-1A or FAD2-1B. The TAL effector nuclease can comprise a monomer that binds to a sequence set forth in any of SEQ ID NOs: 27-34. The TAL effector nuclease can comprise a pair of monomers selected from the group consisting of SEQ ID NOs: 27 and 28, 29 and 30, 31 and 32, and 33 and 34.

[0015] In some embodiments, each second target gene is a gene of the Glycine max FAD2 gene family. In some embodiments, the first target gene is an allele of the Glycine max FAD2-1A gene. In some embodiments, the first target gene is an allele of the Glycine max FAD2-1B gene. In some embodiments, the second target gene is an allele of the Glycine max FAD2-1A gene. In some embodiments, the second target gene is an allele of the Glycine max FAD2-1B gene.

[0016] In some embodiments, the first half-site sequence is within SEQ ID NO:25 or 26.

[0017] In some embodiments, the composition comprises two second nucleic acids each encoding a second transcription activator-like (TAL) effector nuclease monomer capable of binding to a second half-site sequence, wherein the second half-site sequence is SEQ ID NO: 17 or SEQ ID NO: 19.

[0018] In another aspect, the disclosure features a method of simultaneously introducing mutations into two or more genes, the method including contacting a population of cells having the two or more genes with a composition according to one or more of the above embodiments.

[0019] In another aspect, the disclosure features a plant, plant part, or plant cell obtained by the above method. The plant, plant part, or plant cell can be a soybean plant, plant part, or plant cell. The soybean plant part or plant cell can be selected from the group consisting of a cotyledon cell, a seed, an embryo, an embryogenic callus cell, and a pollen cell.

[0020] In another aspect, the disclosure features a soybean oil composition comprising soybean oil produced by the soybean plant, plant part, or plant cell described in the above embodiments, wherein the soybean oil has one or more of an increased oleic acid content, a decreased linoleic acid content, and a decreased linolenic acid content compared to oil produced from a corresponding soybean plant, plant part, or plant cell lacking mutations in the two or more genes.

[0021] In another aspect, the disclosure features a soybean plant, plant part, or plant cell having one or more mutations that reduce expression of at least one of the FAD2-1A and FAD2-1B genes, wherein the plant, plant part, or plant cell produces oil having an increased oleic acid content compared to oil produced from a corresponding soybean plant, plant part, or plant cell lacking the one or more mutations, wherein the at least one mutation is induced by a rare-cutting endonuclease capable of binding to a nucleic acid sequence from the group set forth in SEQ ID NOs:27-34, or a functional variant thereof.

[0022] In another aspect, the disclosure features a method for generating a soybean plant having a mutation that reduces expression of at least one of the FAD2-1A and FAD2-1B genes, the method including: (a) contacting a population of soybean plant cells from a soybean plant having functional FAD2-1A and FAD2-1B genes with one or more nucleic acid sequences encoding a rare-cutting endonuclease capable of binding to a nucleic acid sequence from the group set forth in SEQ ID NOs: 27-34, or a functional variant thereof; (b) selecting cells from the population having reduced expression of the FAD2-1A or FAD2-1B gene; and (c) regenerating the selected plant cells into soybean plants.

[0023] In another aspect, the disclosure features a soybean oil composition, including soybean oil produced by a soybean plant, plant part, or plant cell comprising one or more mutations that reduce expression of at least one of the FAD2-1A and FAD2-1B genes, wherein the soybean oil has one or more of an increased oleic acid content, a decreased linoleic acid content, and a decreased linolenic acid content compared to oil produced from a corresponding soybean plant, plant part, or plant cell lacking the one or more mutations, wherein the one or more mutations comprise a targeted mutation induced by a rare-cutting endonuclease capable of binding to a nucleic acid sequence from the group set forth in SEQ ID NOs:27-34, or a functional variant thereof.

[0024] In another aspect, the disclosure provides a soybean plant, plant part, or plant cell comprising: a first set of mutations in one or more FAD3A alleles and one or more FAD3B alleles, one or more FAD3A alleles and one or more FAD3C alleles, one or more FAD3B alleles and one or more FAD3C alleles, or one or more FAD3A alleles, one or more FAD3B alleles, and one or more FAD3C alleles, wherein the first set of mutations encodes a first transcription activator-like (TAL) effector nuclease monomer capable of binding to a first half-site sequence of a first target gene; and a set of second nucleic acids, each second nucleic acid encoding a second transcription activator-like (TAL) effector nuclease monomer capable of binding to a second half-site sequence of the first target gene and at least one second target gene. and a second set of nucleic acids encoding a first TAL effector nuclease monomer, wherein the first half-site sequence is a conserved sequence, the first half-site sequence and each second half-site sequence are different and separated by a spacer sequence, and the first TAL effector nuclease monomer can form a dimer with each of the second TAL effector nucleases; and mutations in one or more FAD2-1A alleles, one or more FAD2-1B alleles, or one or more FAD2-1A alleles and one or more FAD2-1B alleles, wherein the plant, plant part, or plant cell produces oil having a reduced linolenic acid content, an increased oleic acid content, and a reduced linoleic acid content compared to oil produced from a corresponding soybean plant, plant part, or plant cell lacking the mutations. The plant, plant part, or plant cell can lack the transgene. The plant part can be a seed. The mutations in one or more FAD2-1A alleles and one or more FAD2-1B alleles may be induced by a rare-cutting endonuclease. The rare-cutting endonuclease may be a TAL effector nuclease.The TAL effector nuclease can bind to a sequence set forth in any of SEQ ID NOs: 27 to 34. One or more FAD3A alleles, one or more FAD3B alleles, one or more FAD3C alleles, one or more FAD2-1A alleles, and one or more FAD2-1B alleles can be mutated.

[0025] In another aspect, the disclosure provides a method for generating a soybean plant comprising a mutation that reduces expression of at least two of the FAD3A gene, the FAD3B gene, and the FAD3C gene, comprising: (a) dissolving a population of soybean plant cells from a soybean plant having functional FAD3A, FAD3B, and FAD3C genes in a composition comprising a first nucleic acid encoding a first transcription activator-like (TAL) effector nuclease monomer capable of binding to a first half-site sequence of a first target gene; and a set of second nucleic acids, each second nucleic acid encoding a second transcription activator-like (TAL) effector nuclease monomer capable of binding to a second half-site sequence of the first target gene and to at least one second target gene; and a second set of nucleic acids, wherein the first half-site sequence is a conserved sequence, the first half-site sequence and each second half-site sequence are different and separated by a spacer sequence, the first TAL effector nuclease monomer can form a dimer with each of the second TAL effector nucleases, the first target gene is the FAD3A gene, and at least one second target gene is selected from the FAD3B gene and the FAD3C gene; (b) selecting from the population cells having reduced expression of the first target gene and at least one second target gene; and (c) regenerating the selected plant cells into soybean plants. The first or second monomer may be capable of binding to a nucleic acid sequence selected from the group set forth in SEQ ID NOs: 4-19, or a functional variant thereof. [Brief explanation of the drawings]

[0026] The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings.

[0027] [Figure 1A] 1 shows an alignment of exemplary multiple TALENs targeting the FAD3 gene family of Glycine max according to one or more embodiments of the present disclosure. (A) Alignment of GmFAD3_T01-L1, GmFAD3_T01-R1, GmFAD3_T02-L1, and GmFAD3_T02-R1 (SEQ ID NOs: 45-48, respectively, in order of appearance). [Figure 1B] (B) Alignment of GmFAD3_T03-L1, GmFAD3_T03-R1, GmFAD3_T04-L1, and GmFAD3_T04-R1 (SEQ ID NOs: 49 to 52, respectively, in order of appearance). [Figure 1C] (C) Alignment of GmFAD3_T05-L1, GmFAD3_T05-R1, GmFAD3_T06-L1, and GmFAD3_T07-L1 (SEQ ID NOs: 53 to 56, respectively, in order of appearance). [Figure 1D] (D) Alignment of GmFAD3_T08-L1, GmFAD3_T08-R1, GmFAD3_T09-L1, and GmFAD3_T09-R1 (SEQ ID NOs: 57 to 60, respectively, in order of appearance). [Figure 2A] 1 shows an alignment of exemplary TALENs targeting the FAD2 gene family of Glycine max according to one or more embodiments of the present disclosure: (A) GmFAD2_T01-L1 and GmFAD2_T01-R1 ​​(SEQ ID NOs: 61-62, respectively, in order of appearance). [Figure 2B] (B) Alignment of GmFAD2_T02-L1, GmFAD2_T02-R1, GmFAD2_T03-L1, GmFAD2_T03-R1, GmFAD2_T04-L1, GmFAD2_T04-R1, GmFAD2_T05-L1, and GmFAD2_T05-R1 (SEQ ID NOs: 63 to 65, respectively, in order of appearance). [Figure 2B-1]An expanded portion of the diagram in Figure 2B is shown as diagram B-1 for expanded sequences. In order of appearance, Figure 2B-1 discloses residues 1-44 of SEQ ID NOs: 63-65. [Figure 2B-2] An enlarged portion of the diagram in Figure 2B is shown as diagram B-2 for expanded sequences. Figure 2B-2 discloses residues 45-103 of SEQ ID NOs: 63-65, all in order of appearance, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0028] Provided herein are compositions and methods for using specifically engineered slow-cutting endonuclease technology to target a single gene sequence or simultaneously target multiple similar gene sequences, for example, genes that are part of a gene family, such as the FAD3 gene family in plants.

[0029] The low-frequency-cutting endonuclease can be a TAL effector nuclease, meganuclease, engineered homing endonuclease, zinc finger nuclease (ZFN), or clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system (CRISPR / Cas9). Low-frequency-cutting endonucleases can be natural or engineered proteins with endonuclease activity directed toward nucleic acid sequences with recognition sequences (target sequences) of approximately 12-40 base pairs (bp) in length, or longer (e.g., 14-40, 15-36, or 16-32 in length; see, e.g., Baker, Nature Methods 9:23-26, 2012). Typical low-frequency-cutting endonucleases cleave within their recognition site, leaving a staggered four-nucleotide (nt) cut with a 3' or 5' OH overhang. In some embodiments, the rare-cutting endonuclease is a meganuclease, such as a wild-type or variant homing endonuclease (e.g., a homing endonuclease belonging to the dodecapeptide family (see WO2004 / 067736)).

[0030] The low-frequency-cutting endonuclease may be based on the RNA-guided Cas9 nuclease from the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system. This system allows for cleavage of DNA sequences adjacent to a short sequence motif called a protospacer adjacent motif (PAM). Cleavage is achieved by engineering a specific crRNA that is complementary to the target sequence. The Cas9 endonuclease forms a complex with the CRISPR RNA (crRNA). The dual trans-activating crRNA (tracrRNA): the crRNA structure acts as a guide RNA (gRNA) that directs the Cas9 endonuclease to the target sequence. The PAM motif in the sequence found in the FAD2-1 or FAD3 gene family allows for the design of specific crRNAs to introduce mutations or inactivate one or more targeted genes in plant cells transfected with the crRNA, heterologously expressing the Cas9 endonuclease.

[0031] In some embodiments, the rare-cutting endonuclease is a fusion protein containing a DNA-binding domain and a catalytic domain with cleavage activity. TALE nucleases and ZFNs are examples of fusions of a DNA-binding domain with the catalytic domain of the endonuclease FokI. Custom-made TALE nucleases are commercially available under the trade name TALEN™ (Cellectis, Paris, France). The specificity of transcription activator-like (TAL) effectors depends on the effector variable repeat. Polymorphisms primarily exist at repeat positions 12 and 13, known as repeat variable dinucleotides (RVDs). The RVDs of TAL effectors correspond to nucleotides in their target sites in a direct, linear manner, with one RVD per nucleotide, with some degeneracy and no apparent context dependency. This mechanism for protein-DNA recognition enables target site prediction for new target-specific TAL effectors, as well as target site selection and engineering of new TAL effectors with binding specificity to selected sites.

[0032] The TAL effector DNA binding domain can be fused to other sequences, such as endonuclease sequences, resulting in a chimeric endonuclease that targets a specific selected DNA sequence, leading to subsequent cleavage of the DNA at or near the targeted sequence. Such cleavage (double-strand break) in DNA can induce mutations in wild-type DNA sequences, for example, through NHEJ or homologous recombination. TALE nucleases can be used to facilitate site-specific mutagenesis in complex genomes, knocking out or otherwise altering gene function with great precision and high efficiency.

[0033] Methods for selecting endogenous target sequences and generating TALE nucleases that target such sequences can be performed as described in the art. See, for example, PCT Publication No. WO2011 / 072246 (incorporated by reference). In some embodiments, software can be used that specifically identifies TALE nuclease recognition sites.

[0034] Some endonucleases (e.g., FokI) function as dimers, thereby enhancing target specificity. When two TALE nuclease recognition sites are in close proximity, the inactive monomers can combine to create a functional enzyme that cleaves DNA. By requiring DNA binding to activate the nuclease, highly site-specific restriction enzymes can be created.

[0035] The TALENs of the present disclosure include TALENs that can target multiple genes that share a common (e.g., conserved) sequence. The TALENs of the present disclosure include a common first TAL effector nuclease monomer (e.g., a left half TALEN (left HT) or a right half TALEN or (right HT)) and a set of second TAL effector nuclease monomers (e.g., two, three, four, or more second HTs), where the common HT can form a dimer with any of the second HTs to enable targeting of all intended sequences. As used herein, the terms "TAL effector nuclease monomer," "half TALEN," and "HT" are used interchangeably. These first TAL effector nuclease monomers and sets of second TAL effector nuclease monomers can be used in combination with TALENs engineered to target a single sequence, such as the sequence of the FAD2-1 gene (e.g., at least one of FAD2-1A and FAD2-1B).

[0036] Use of the disclosed compositions and methods includes aligning gene sequences of interest to determine regions of approximately conserved identity between sequences. In some embodiments, a common first TAL effector nuclease monomer (e.g., left HT or right HT) targets a sequence that is about 100% conserved, about 95% conserved, about 90% conserved, about 85% conserved, or about 80% conserved across all genes targeted by the disclosed compositions and methods.

[0037] For example, in one embodiment, 100% conserved targeting region is selected for the left HT binding domain (half-site sequence).For the right HT binding domain, a unique HT is designed for each non-conserved sequence to enable targeting.In this non-limiting example, gene 1 is targeted by left HT#1 and right HT#1, gene 2 is targeted by left HT#1 and right HT#2, gene 3 is targeted by left HT#1 and right HT#3, etc.

[0038] Similarly, for example, in another embodiment, 100% conserved targeting region is selected for right HT binding domain.For left HT binding domain, each non-conserved sequence is designed with a unique HT to enable targeting.In this non-limiting example, gene 1 is targeted by left HT#1 and right HT#1, gene 2 is targeted by left HT#2 and right HT#1, gene 3 is targeted by left HT#3 and right HT#1, etc.

[0039] In some embodiments, the genes targeted by the compositions and methods of the present disclosure can encode the same protein or different proteins (e.g., a common HT targets a sequence that is conserved or identical in all of the genes). In some embodiments, the genes targeted by the compositions and methods of the present disclosure are alleles of the gene.

[0040] In some embodiments, the targeted gene is the FAD3A, FAD3B, or FAD3C gene. In some embodiments, the targeted gene is SEQ ID NO: 1-3. In some embodiments, the methods and compositions disclosed herein may target, e.g., introduce mutations, in any two of the FAD3A, FAD3B, and FAD3C genes. In some embodiments, the methods and compositions disclosed herein may target, e.g., introduce mutations, in all three of the FAD3A, FAD3B, and FAD3C genes. One or more mutations may be present in the coding or non-coding sequence of the FAD3A, FAD3B, or FAD3C gene. Genomic sequences of native soybean genes can be found in the Soybase Database (www.soybase.org). In some embodiments, the compositions and methods disclosed herein comprise a transcription activator-like (TAL) effector nuclease monomer capable of binding to a sequence selected from SEQ ID NO: 4-19, or a sequence within any one of SEQ ID NO: 1-3.

[0041] In some embodiments, the methods and compositions disclosed herein can target, for example, introduce mutations in either the FAD2-1A or FAD2-1B gene. In some embodiments, the targeted gene is the FAD2-1A or FAD2-1B gene. One or more mutations can be present in the coding sequence or non-coding sequence of the FAD2-1A or FAD2-1B gene. The endogenous exemplary genomic sequences of soybean FAD2-1A and FAD2-1B genes are shown in SEQ ID NOs: 23 and 24, respectively. In some embodiments, the targeted gene is SEQ ID NO: 23 or 24, or a functional variant thereof that has at least about 80%, for example, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 23 or 24. Sometimes, the targeted sequence is within the coding sequence of the FAD2-1A or FAD2-1B gene, such as the coding sequence set forth in SEQ ID NO: 25 or 26. The target sequence can be a sequence within SEQ ID NO: 25 or 26, or a functional variant thereof having at least about 80%, e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 25 or 26.

[0042] The percent sequence identity between any nucleic acid sequence and a sequence referenced by a sequence number (SEQ ID NO:) can be determined by conventional methods. In one example, a nucleic acid sequence is compared to a sequence set forth in a sequence number using the BLAST 2 Sequences (Bl2seq) program from the standalone version of BLASTZ, including BLASTN version 2.0.14. If the two compared sequences share homology, the designated output file will present the regions of homology as aligned sequences. If the two compared sequences do not share homology, the designated output file will not present aligned sequences. Once aligned, the number of matches is determined by counting the number of positions where identical nucleotide residues are represented in both sequences. The percent sequence identity is determined by dividing the number of matches by either the length of the sequence set forth in the identified sequence (e.g., SEQ ID NO: 1) or by the segmented length (e.g., 100 consecutive nucleotides from the sequence set forth in the identified sequence), followed by multiplying the resulting value by 100. The percent sequence identity value is rounded to the nearest tenth. For example, the targeted gene may have a coding sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20, 21, 22, 25, or 26.

[0043] In some embodiments, the compositions and methods disclosed herein include a transcription activator-like (TAL) effector nuclease monomer capable of binding to a sequence within the genomic or coding sequence of at least one of the FAD2-1A and FAD2-1B genes. For example, the TALEN monomer can be selected from one of SEQ ID NOS: 27-34, including pairs of monomers selected from the group consisting of SEQ ID NOS: 27 and 28, 29 and 30, 31 and 32, 33 and 34, and 35 and 36, or functional variants of these sequences. Functional variants include, for example, sequences with one or more nucleotide substitutions, deletions, or insertions, but retaining the desired activity. Functional variants can be generated by any of several methods available to those skilled in the art, such as site-directed mutagenesis, induced mutations, identification as allelic variants, cleavage using restriction enzymes, etc.

[0044] The composition disclosed herein comprises the TALEN of the present disclosure and the nucleic acid that encodes the TALEN of the present disclosure.Therefore, in some embodiments, the composition provided herein comprises: a first nucleic acid that encodes HT that targets the common sequence that is conserved across all genes that are targeted by the composition; and a set of second nucleic acids that encode a plurality of second HTs that target second sequences, wherein the HT that targets common sequence can form dimers with each of the second HTs.

[0045] TALEN gene editing is based on gene-specific targeting of two half-TALEN proteins containing a DNA recognition domain and a FokI endonuclease domain encoded in a plasmid containing an appropriate promoter, terminator, and other non-coding sequences necessary for expression in recipient cells. FokI is only active in dimeric combinations, and two half-TALENs are required to enable the endonuclease DNA cleavage required for TALEN gene editing.

[0046] The compositions of the present disclosure can be delivered to eukaryotic cells, mammalian cells, plant cells, or prokaryotic cells. Introduction into cells can be achieved by several methods known to those skilled in the art. Common methods for introducing gene editing TALENs include transformation using Agrobacterium spp. carrying a plasmid containing a TALEN gene cassette, particle bombardment using a plasmid or mRNA encoding a TALEN sequence cassette, and PEG-mediated transformation using a plasmid or mRNA encoding a TALEN sequence cassette. In some embodiments, the compositions of the present disclosure can be used to generate a set of half-TALEN proteins encoded by the compositions. The half-TALEN proteins can be isolated and then introduced into target cells, such as plant cells, by several methods, including, for example, PEG-mediated transfection (such as the technique described in Luo S. et al., "Non-transgenic Plant Genome Editing Using Purified Sequence-Specific Nucleases," Mol Plant. 2015 Sep;8(9):1425-7).

[0047] The plant cell may be from a soybean plant. The terms "soybean plant" or "plant part" are used broadly to include soybean plants at any developmental stage, or parts of soybean plants, including plant cuttings, plant cells, plant cell cultures, plant organs, plant seeds, and plantlets. A plant cell is a structural and physiological unit of a plant, including a protoplast and cell wall. A plant cell may be in the form of an isolated single cell or an aggregate of cells, such as a friable callus or cultured cell, or may be part of a more highly organized unit, such as a plant tissue, plant organ, or plant. Thus, a plant cell may be a protoplast, a gamete-producing cell, or a cell or collection of cells that can regenerate into a whole plant. Therefore, a seed containing multiple plant cells and capable of regenerating into a whole plant is considered a plant cell for purposes of this disclosure. A plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells organized into a structural or functional unit. Particularly useful parts of a plant include harvestable parts and parts useful for propagating progeny plants. Harvestable parts of a plant can be any useful part of the plant, e.g., flowers, pollen, seedlings, leaves, stems, pods, seeds, roots, nodules, etc. Parts of a plant that are useful for propagation include, e.g., seeds, pods, cuttings, seedlings, rhizomes, etc. "Seed" refers to any plant structure formed by the successive differentiation of the plant's ovule following its normal point of maturity at anthesis, whether it is formed in the presence or absence of fertilization, and whether the seed structure is fertile or sterile.

[0048] Low-frequency-cutting endonucleases (e.g., TALENs) can be encoded by nucleic acid sequences for expression in plant cells or can be introduced as proteins. Nucleic acids encoding sequence-specific nucleases can be introduced into soybean plants by Agrobacterium-mediated transformation of plant parts or plant cells (e.g., leaves, stems, petioles, internodal explants, calli, or protoplasts) with T-DNA encoding the low-frequency-cutting endonuclease, biolistic transformation of plant parts or plant cells with one or more nucleic acids encoding the low-frequency-cutting endonuclease, cell-penetrating peptide-mediated transformation, and / or polyethylene glycol (PEG)-mediated transformation. For example, protoplasts can be isolated from surface-sterilized leaves and transformed with one or more plasmids encoding low-frequency-cutting endonucleases in the presence of PEG. Transformation efficiency can be monitored by the delivery of a detectable marker, such as a YFP plasmid, which can be visualized using fluorescence microscopy or flow cytometry. After PEG-mediated transformation, the protoplasts can be cultured using methods and media known to those skilled in the art for protoplast culture. After a suitable length of time in culture, callus from protoplasts identified as mutants can be propagated, transferred to shoot induction medium, and then (once roots have formed) transferred to soil and grown to maturity for seed production.

[0049] The low-frequency-cutting endonuclease can be delivered to soybean plants using methods for transient expression or by using methods for stable integration into the host genome. To transiently deliver the sequence-specific nuclease, transformed soybean plant parts or plant cells (e.g., using the methods described above) can be placed on a regeneration medium that does not contain a selection agent, and soybean plants can be regenerated. The regenerated plants can be screened to identify those containing the induced mutation. To stably integrate the genome manipulation reagent into the host genome, the nucleic acid encoding the low-frequency-cutting endonuclease can be co-delivered with a nucleic acid encoding a plant selection marker. The selection marker can be carried on the same vector as the low-frequency-cutting endonuclease or can be delivered as a separate vector. After transformation, the soybean plant parts or plant cells can be placed on a regeneration medium containing the appropriate selection agent. The transformed plant cells can be regenerated into transgenic soybean plants. In some cases, the soybean plants do not contain a transgene or any exogenous DNA (e.g., T-DNA). Progeny free of exogenous DNA (e.g., sequence-specific nuclease sequences) can be generated by segregation, which can stabilize induced mutations and satisfy biosafety concerns.

[0050] In the above-mentioned method, the low-frequency cutting endonuclease can be co-delivered to plant cells with the plasmid encoding one or more exonuclease proteins to increase the efficiency of sequence-specific nuclease-induced mutagenesis.Such exonucleases include, but are not limited to, members of the TREX (3' ​​repair exonuclease) family of exonucleases, such as TREX2.

[0051] The present disclosure provides materials and methods for editing one or more genes encoding FAD2 or FAD3 using a rare-cutting endonuclease (e.g., a TALE nuclease) to produce soybean plants and related products (e.g., seeds and plant parts) that are particularly suitable for providing high oleic and / or low linoleic or linolenic fatty acid oils. A method of using the compositions of the present disclosure can include contacting a population of soybean plant cells (e.g., protoplasts) that express one or more of the FAD2-1A, FAD2-1B, FAD3A, FAD3B, and FAD3C genes with a rare-cutting endonuclease engineered to induce targeted mutations in one or more genes, and selecting cells from the population that have mutations that reduce expression of one or more genes. In addition, the method may include isolating genomic DNA containing at least a portion of one or more targeted loci (e.g., FAD2-1A, FAD2-1B, FAD3A, FAD3B, or FAD3C loci) from the plant cell. In some cases, the method includes culturing the plant cell containing the mutation to generate one or more plant lines. Parts of the soybean plant, such as seeds, synthesize and accumulate fatty acids, which can be identified and quantified. For example, analysis of the fatty acid composition of the plant parts, as described in the Examples, can confirm that the mutation leads to increased levels of oleic acid, decreased levels of linoleic acid, decreased levels of linolenic acid, or a combination of these characteristics in the generated plant line compared to soybean plants that do not contain the mutation.

[0052] In certain embodiments, the expression of targeted genes is reduced by induced mutation.Reducing the expression of genes in plants, plant parts, or plant cells includes inhibiting, disrupting, knocking out, or knocking down genes, so that the transcription of genes and / or the translation of encoded polypeptides is reduced compared to corresponding control plants, plant cells, or populations of plants or plant cells in which the expression of genes or polypeptides is not inhibited, disrupted, knocked out, or knocked down.Reduction encompasses any reduction in expression level (e.g., 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or even 100% reduction) compared to corresponding control plants, plant cells, or populations of plants or plant cells.In some embodiments, reducing expression by 50% or more may be particularly useful. Expression levels can be measured using methods such as reverse transcription polymerase chain reaction (RT-PCR), Northern blotting, dot blot hybridization, in situ hybridization, nuclear run-on and / or nuclear run-off, RNase protection, or immunological and enzymatic methods such as ELISA, radioimmunoassay, and Western blotting.

[0053] The low-cutting endonuclease can be introduced into a population of cells via a nucleic acid (e.g., a vector or mRNA) encoding a sequence-specific nuclease, or as a protein. For example, a nucleic acid encoding a TALE nuclease targeting a conserved nucleotide sequence present in one or more FAD2 and FAD3 genes can be used to transform soybean plant cells or plant parts (e.g., protoplasts) and express them therein. In some cases, the TALE nuclease protein can be introduced into soybean plant cells or plant parts (e.g., protoplasts). These cells or plant parts, or plant cell lines or plant parts generated from these cells, can be analyzed to determine whether mutations have been introduced at the target site using next-generation sequencing technology (e.g., 454 pyrosequencing or Illumina sequencing). The template for sequencing can be the targeted gene amplified by PCR using primers homologous to the conserved nucleotide sequence. Cells can also be analyzed for any off-target mutations.

[0054] In some embodiments, one or more nucleic acids encoding multiple TALE nucleases can be used to transform soybean plant cells or plant parts (e.g., protoplasts) and express them therein. These TALE nucleases can be introduced into the plant cells or parts as proteins. The expressed or introduced TALE nucleases can generate double-strand breaks (DSBs) on the same chromosome, resulting in deletion of the intervening sequence. These cells or plant parts, or plant cell lines or plant parts generated from these cells, can be analyzed for targeted gene loss. Deletions of loci containing the FAD2-1 or FAD3 gene family can be analyzed qualitatively or quantitatively. For example, a first primer can be designed to be homologous to the sequence upstream of the first TALE nuclease target site, and a second primer can be designed to be complementary to the sequence downstream of the second TALE nuclease target site. If a targeted deletion occurs, a PCR product is obtained. Deletion of the locus containing the targeted gene can also be analyzed by qPCR. A lower copy number of the targeted gene relative to the unmodified control may indicate the presence of an intended deletion. In the T7E1 assay, genomic DNA can be isolated from pooled calli, and sequences adjacent to the TALE nuclease recognition site of the targeted gene can be PCR amplified. The amplified products can then be denatured and reannealed. If the reannealed fragments form a heteroduplex, T7 endonuclease I cleaves at the site of the mismatch. The digested products can be visualized by gel electrophoresis to quantify the mutagenic activity of the TALE nuclease.

[0055] Methods for contacting a population of soybean plant cells to deliver a sequence-specific nuclease may include Agrobacterium-mediated transformation of plant parts or plant cells (e.g., leaves, stems, petioles, internodal explants, callus, or protoplasts) with a T-DNA encoding the sequence-specific nuclease, biolistic transformation of plant parts or plant cells with one or more nucleic acids encoding the sequence-specific nuclease, and / or cell-permeable peptide-mediated transformation of plant parts or plant cells with purified sequence-specific nuclease or nucleic acids (RNA or DNA) encoding the sequence-specific nuclease.

[0056] Polyethylene glycol (PEG)-mediated transformation can be used to deliver sequence-specific nucleases. For example, protoplasts can be isolated from surface-sterilized leaves and transformed with a plasmid encoding one or more sequence-specific nucleases in the presence of PEG. Transformation efficiency can be monitored by the delivery of a detectable marker, such as a YFP plasmid, which can be visualized using fluorescence microscopy or flow cytometry. After PEG-mediated transformation, protoplasts can be cultured using methods and media known to those skilled in the art of protoplast culture. After a suitable length of time in culture, calli derived from protoplasts identified as mutants can be propagated and transferred to shoot induction medium, and then (once roots have formed) transferred to soil and grown to maturity for seed production.

[0057] In some cases, soybean plants contain multiple mutations directed at altering the fatty acid composition of oil produced by the plant, plant part, or plant cell. Within the scope of the present disclosure are embodiments featuring soybean plants, plant parts, or plant cells with mutations that regulate the expression of FAD2 and FAD3 proteins, wherein the plant, plant part, or plant cell produces oil with increased oleic acid and decreased linoleic and linolenic fatty acid content compared to oil produced from a corresponding soybean plant, plant part, or plant cell lacking one or more mutations. Targeted FAD2-1A, FAD2-1B, or FAD3A / B / C expression mutants can be imparted to any soybean line, species, or cultivar of interest, including, but not limited to, those with a genetic predisposition for higher oleic acid levels and lower linoleic or linolenic acid levels. In some embodiments, targeted gene editing or other genetic modification can be imparted to germplasm or other plant tissues that already possess a trait (e.g., a genetic predisposition) for higher oleic acid levels and lower linoleic or linolenic acid levels.

[0058] Mutant soybean lines may contain mutations that provide or alter the expression of proteins other than FAD2 and FAD3. Combination effects may include the use of multiple separate nucleic acid constructs or transformation events. For example, multiple constructs such as those described above can be introduced into plant cells by the same or different methods, including the introduction of such traits by including two transcription cassettes in a single transformation vector, co-transformation of two expression constructs, re-transformation with a second expression construct, or by crossing transgenic plants through traditional plant breeding methods, as long as the resulting product is a plant that has both characteristics integrated into its genome.

[0059] Traditional breeding techniques can be combined with the targeted approaches described above. In some cases, the soybean plants that receive the constructs are elite lines possessing one or more specific agronomically important traits, such as those that result in increased biomass production, increased food production, improved food quality, pest resistance, vigor, developmental time (time to harvest), enhanced nutrient content, novel growth patterns, flavor or color, salt, heat, drought and cold tolerance, etc.

[0060] The transformed soybean plants of the present disclosure can be used in plant breeding programs to create new and useful lines and varieties. Breeding can be carried out via known procedures. DNA fingerprinting, SNP, or similar techniques can be used in marker-assisted selection (MAS) breeding programs to transfer or breed mutations that regulate the expression of one or more of the FAD2 and FAD3 genes into other soybean plants. In some embodiments, such methods include creating a cross between the soybean mutant and a second soybean plant to produce an F1 plant, or a species that can be crossed with the mutant. The method can further include backcrossing the F1 plant to a second soybean plant and repeating the backcrossing step to generate a near-isogenic line with the mutation integrated into the genome of the second soybean plant, wherein the near-isogenic line derived from the second plant with the integrated mutation has an altered fatty acid profile (e.g., higher oleic acid levels, lower linoleic acid levels, and / or lower linolenic acid levels). Such methods can be facilitated by molecular markers or TILLING®. Thus, the mutant soybean plants of the present disclosure can be used to generate new and useful lines and varieties.

[0061] In some embodiments, the methods described herein can be used to produce soybean varieties with oils that have superior stability and performance, resulting in part from the oil's fatty acid profile. Commercial soybean oil is primarily composed of five fatty acids: palmitic acid (10%), stearic acid (4%), oleic acid (18%), linoleic acid (55%), and linolenic acid (13%). Soybean oil with a lower linolenic acid content may increase its oxidation and frying stability. Such oils may be healthier, particularly since they do not require partial hydrogenation of polyunsaturated fatty acids for stabilization. Partial hydrogenation produces trans fatty acids, the consumption of which is associated with an increased risk of heart disease. The soybean varieties disclosed herein may have oils with reduced linolenic acid content, increased oleic acid content, or reduced linoleic acid content compared to soybean varieties lacking the above mutations. For example, soybean varieties of the present disclosure with stacked mutations within the FAD2-1 and FAD3 gene families may have oil with linolenic and linoleic acid levels of less than 3% and oleic acid levels of greater than 65%.

[0062] As used herein, the term "gene" refers to a nucleic acid sequence comprising a promoter region associated with expression of a gene product. A "gene" also encompasses intron and exon regions associated with expression of the gene product, as well as 5' or 3' untranslated regions associated with expression of the gene product.

[0063] "Endogenous gene" refers to a nucleic acid molecule that contains a sequence of the wild-type sequence occurring in a wild-type plant, or a sequence with a percent identity (e.g., a sequence with at least 90% identity) that allows the encoded product to retain function, and can be obtained from a plant or plant part of a cell, or can be produced synthetically. Further embodiments provide that the sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0064] "FAD2" refers to the fatty acid desaturase 2 (FAD2) protein, which is encoded by three FAD2 desaturase genes in the soybean genome; however, FAD2-1A (Glyma10g42470) and FAD2-1B (Glyma20g24530) are highly expressed during peak oil synthesis and are the major genetic determinants of oleic acid and linoleic acid levels in soybean seeds. FAD2 catalyzes the conversion of oleic acid to linoleic acid.

[0065] "FAD3" refers to the fatty acid desaturase 3 (FAD3) enzyme, which is produced by a family of genes consisting of FAD3A (Glyma14g37350), FAD3B (Glyma02g39230), and FAD3C (Glyma18g06950), and catalyzes the conversion of linoleic acid to linolenic acid.

[0066] The term "functional variant" refers to a catalytically active variant of a described protein or protein domain, a nucleotide sequence encoding a variant of a protein or protein domain that performs the same function as a described SEQ ID NO:, or a nucleotide sequence variant that mediates the same activity (e.g., TALEN-mediated cleavage) as a described SEQ ID NO:.

[0067] As used herein, the term "about" indicates that the associated value, unless otherwise indicated, can be modified by plus or minus five percent (+ / - 5%) to remain within the scope of the disclosed embodiments.

[0068] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed herein to include the group as modified, and thus fulfills the written description of all Markush groups used in the appended claims.

[0069] Unless otherwise indicated, nucleic acids or oligonucleotides are written left to right in 5' to 3' orientation.

[0070] The words "herein," "above," and "below," and words of similar import, when used in this application, refer to this application as a whole and not to any particular portions of this application, unless otherwise indicated. As used herein, the words "about" and "approximately" include slight variations around the stated value, typically within a standard error range, such as within 10% of the stated value.

[0071] The referenced patents, patent applications, and scientific literature referred to in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0072] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0073] 1. Multiple TALEN design for fatty acid desaturase 3 (FAD3) gene family in soybean. The genomic sequences of Glycine max FAD3A, FAD3B, and FAD3C (SEQ ID NOs: 1-3) were aligned using software such as Geneious (Biomatters Ltd) to identify regions of homology between the genes. GmFAD3_T01-L1 and GmFAD3_T01-R1 ​​(SEQ ID NOs: 4-5) were designed with targeting specificity for FAD3A and FAD3B (SEQ ID NOs: 1-2). GmFAD3_T02-L1 and GmFAD3_T02-R1 (SEQ ID NOs: 6-7) were designed with specificity for FAD3C (SEQ ID NO: 3) (Figure 1A). GmFAD3_T03-L1 and GmFAD3_T03-R1 (SEQ ID NOs: 8-9) were designed with targeting specificity for FAD3A and FAD3B (SEQ ID NOs: 1-2). GmFAD3_T04-L1 and GmFAD3_T04-R1 (SEQ ID NOs: 10-11) were designed with specificity for FAD3C (SEQ ID NO: 3) (Figure 1B). GmFAD3_T05-L1 (SEQ ID NO: 12) was designed with specificity for FAD3A (SEQ ID NO: 1). GmFAD3_T05-R1 (SEQ ID NO: 13) was designed with specificity for FAD3A, FAD3B, and FAD3C (SEQ ID NOs: 1-3). GmFAD3_T06-L1 (SEQ ID NO: 14) was designed with specificity for FAD3B (SEQ ID NO: 2). GmFAD3_T07-L1 (SEQ ID NO: 15) was designed with specificity for FAD3C (SEQ ID NO: 3) (Figure 1C). GmFAD3_T08-L1 and GmFAD3_T08-R1 (SEQ ID NOs: 16-17) were designed with targeting specificity for FAD3A and FAD3B (SEQ ID NOs: 1-2). GmFAD3_T09-L1 and GmFAD3_T09-R1 (SEQ ID NOs: 18-19) were designed with targeting specificity for FAD3C (SEQ ID NO: 3) (Figure 1D).

[0074] 2. Demonstration of nuclease activity for HT couples in soybean protoplasts Half-TAL effector nucleases (half-TALENs or HTs) were synthesized, cloned into plant expression vectors, and transformed into soybean protoplasts using a PEG-mediated protocol to evaluate the activity of individual combinations of half-TALEN pairs. Forty-eight hours after transformation, DNA was extracted from the protoplast populations, and PCR was used to amplify and sequence the target genes, FAD3A, FAD3B, and FAD3C (SEQ ID NOs: 1-3), using Illumina Miseq technology. Analysis was performed to determine non-homologous end joining (NHEJ) frequency as a measure of TALEN nuclease activity. For each TALEN combination, nuclease activity for each of the three target genes was determined. The data are summarized in Table 1. Using two HT effector nucleases, the highest NHEJ frequencies for FAD3A and FAD3B were achieved using the combination of GmFAD3_T08-L1 and GmFAD3_T08-R1 (22.87% and 14.57%). For FAD3C, the highest activity using a two HT combination was observed using GmFAD3_T09-L1 and GmFAD3_T09-R1 (11.32%).

[0075] 3. Demonstration of nuclease activity for multiple HTs in soybean protoplasts The combination of three half-TAL effector nucleases (half-TALEN or HT) with the highest activity at FAD3A, FAD3B, and FAD3C was selected and cloned into a single transformation vector for delivery into soybean protoplasts using a PEG-mediated protocol. DNA was extracted from the protoplast population 48 hours after transformation, as described above, and PCR was used to amplify and sequence the target genes, FAD3A, FAD3B, and FAD3C (SEQ ID NOs: 1-3), to determine NHEJ activity. Delivery of the GmFAD3_T09-L1, GmFAD3_T08-R1, and GmFAD3_T09-R1 combination successfully achieved highly efficient nuclease activity at all three gene targets: FAD3A (16.28%), FAD3B (14.82%), and FAD3C (21.42%). The data are summarized in Table 2.

[0076] [Table 1]

[0077] [Table 2]

[0078] [Table 3]

[0079] After verifying that the TAL effector nuclease pair generated targeted modifications at the endogenous target site, experiments were performed to generate soybean plants with mutations in two or more FAD3A / B / C. The mutant alleles characterized from exemplary soybean plants are shown in SEQ ID NOs: 40-44.

[0080] Seeds from soybean lines homozygous mutant for two or more of the FAD3A / B / C genes were analyzed for fatty acid composition. Briefly, individual soybean seeds were individually ground. DNA was prepared from a portion of the basal tissue and analyzed to confirm the genotype of each seed. Ground tissue from double and triple homozygous knockout seeds was pooled. Fatty acid composition was then determined using fatty acid methyl ester (FAME) gas chromatography (Beuselinck et al., Crop Sci. 47:747-750, 2006) to assess whether seeds with various FAD3A / B / C mutations have altered proportions of linoleic acid, linoleic acid, and oleic acid relative to wild-type seeds.

[0081] 4. Sequence-specific nucleases for mutagenesis of G.Max FAD2-1A and FAD2-1B genes To completely inactivate or knock out alleles of the FAD2-1A and FAD2-1B genes in G. max, we designed sequence-specific nucleases that target the protein-coding region near the start codon. Eight TAL effector nuclease pairs were designed to target the FAD2-1 gene family (Table 4). The pairs GmFAD2_T01-L1 and GmFAD2_T01-R1 ​​(SEQ ID NOs: 27 and 28), GmFAD2_T02-L1 and GmFAD2_T02-R1 (SEQ ID NOs: 29 and 30), GmFAD2_T03-L1 and GmFAD2_T03-R1 (SEQ ID NOs: 31 and 32), and GmFAD2_T04-L1 and GmFAD2_T04-R1 (SEQ ID NOs: 33 and 34) achieved highly efficient nuclease activity at the FAD2-1A and FAD2-1B gene targets (Table 5).

[0082] [Table 4]

[0083] [Table 5]

[0084] After verifying that the TAL effector nuclease pair produced targeted modifications at the endogenous target site, experiments were performed to generate soybean plants with mutations in one or both of FAD2-1A and FAD2-1B. The mutant alleles characterized from exemplary soybean plants are shown in SEQ ID NOs: 37-39.

[0085] Seeds from soybean lines homozygous mutant for either FAD2-1A or FAD2-1B, or homozygous for both FAD2-1A and FAD2-1B, were analyzed for fatty acid composition. The results are shown in Table 6. Briefly, individual soybean seeds were individually ground. DNA was prepared from a portion of the basal tissue and analyzed to confirm the genotype of each seed. Ground tissue from FAD2-1A homozygous, FAD2-1B homozygous, or FAD2-1A / FAD2-1B double homozygous knockout seeds was pooled. Fatty acid composition was then determined using fatty acid methyl ester (FAME) gas chromatography (as described above) to assess whether seeds with various FAD2-1 mutations had altered proportions of linoleic acid and oleic acid relative to wild-type seeds.

[0086] 5. Combination of FAD2 and FAD3 mutations Plants containing combinations of mutations that knock out the activity of one or more FAD2 and FAD3 genes or proteins were produced by either using TAL effector endonucleases to target one or more genes, or by crossbreeding plants with mutations in the FAD2 and / or FAD3 genes.A series of plants containing combinations of mutations were produced, including plants with two, three, four, or five genes knocked out.The seed oil composition of greenhouse-grown plants was evaluated by FAME.The results are shown in Table 6.

[0087] [Table 6] JPEG0007815114000007.jpg222149 JPEG0007815114000008.jpg219149 JPEG0007815114000009.jpg220149

[0088] The fatty acid profiles show significant changes in the levels of several fatty acids in the oils from the mutant plants compared to those from the WT plants. The levels of linolenic acid, linoleic acid, and palmitic acid decreased, while the levels of eicosenoic acid, oleic acid, and stearic acid increased. In the oils of some mutants (e.g., samples soybean 31-50 and 71-75), the level of erucic acid increased relative to that of the WT plants.

[0089] While exemplary embodiments have been illustrated and described, it will be understood that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

1. 1. A composition comprising: a first nucleic acid encoding a first transcription activator-like (TAL) effector nuclease monomer capable of binding to conserved sequences of two or more targets; a set of second nucleic acids, wherein each second nucleic acid encodes a second TAL effector nuclease monomer capable of binding to a second sequence of one of said two or more targets and capable of forming a dimer with said first TAL effector nuclease monomer, the set of second nucleic acids comprising at least two nucleic acids; the conserved sequence is different from each of the second sequences, and each of the second sequences is different from each other; The two or more targets comprise a spacer sequence between the conserved sequence and each of the second sequences.

2. 2. The composition of claim 1, wherein when the first TAL effector nuclease monomer binds to the conserved sequence and the second TAL effector nuclease monomer binds to each of the second sequences, each dimer can cleave each of the two or more targets in a living cell.

3. The composition of claim 1 or 2, wherein the conserved sequence is a 100% conserved sequence.

4. The composition of any one of claims 1 to 3, wherein the spacer sequence is 15 to 18 nucleotides in length.

5. The composition of any one of claims 1 to 4, wherein the first nucleic acid comprises a FokI endonuclease domain.

6. The composition of any one of claims 1 to 5, wherein each second nucleic acid comprises a FokI endonuclease domain.

7. The composition of any one of claims 1 to 6, wherein the first nucleic acid is in a vector.

8. The composition of any one of claims 1 to 7, wherein each second nucleic acid is in a vector.

9. The composition of any one of claims 1 to 8, wherein the set of the first nucleic acid and the second nucleic acid is in a single vector.

10. The composition of any one of claims 1 to 9, wherein the first nucleic acid comprises mRNA.

11. The composition of any one of claims 1 to 10, wherein each second nucleic acid comprises mRNA.

12. The composition of any one of claims 1 to 11, wherein the set of the first nucleic acid and the second nucleic acid comprises mRNA.

13. The composition of any one of claims 1 to 12, wherein the set of second nucleic acids comprises three or more second nucleic acids.

14. The composition of any one of claims 1 to 13, wherein the set of second nucleic acids comprises four or more second nucleic acids.

15. 15. The composition of any one of claims 1 to 14, wherein a first of the two or more targets is a gene of the FAD3 gene family of Glycine max.

16. 16. The composition of any one of claims 1 to 15, wherein the second of the two or more targets is a gene of the FAD3 gene family of Glycine max.

17. 17. The composition of any one of claims 1 to 16, wherein a first of the two or more targets is an allele of the Glycine max FAD3A gene.

18. 17. The composition of any one of claims 1 to 16, wherein a first of the two or more targets is an allele of the Glycine max FAD3B gene.

19. 17. The composition of any one of claims 1 to 16, wherein a first of the two or more targets is an allele of the Glycine max FAD3C gene.

20. 20. The composition of any one of claims 17 to 19, wherein the second of the two or more targets is an allele of the Glycine max FAD3A gene.

21. 20. The composition of any one of claims 17 to 19, wherein the second of the two or more targets is an allele of the Glycine max FAD3B gene.

22. 20. The composition of any one of claims 17 to 19, wherein the second of the two or more targets is an allele of the Glycine max FAD3C gene.

23. The composition of any one of claims 1 to 22, wherein the conserved sequence is SEQ ID NO:

18.

24. 24. The composition of any one of claims 1 to 23, further comprising one or more rare-cutting endonucleases that target alleles of the FAD2-1 gene family of Glycine max.

25. 25. The composition of claim 24, wherein the one or more rare-cutting endonucleases are TAL effector nucleases that target FAD2-1A or FAD2-1B.

26. 26. The composition of claim 25, wherein the TAL effector nuclease comprises a monomer that binds to a sequence set forth in any of SEQ ID NOs: 27-34.

27. 26. The composition of claim 25, wherein the TAL effector nuclease comprises a pair of monomers that bind to a sequence selected from the group consisting of SEQ ID NOs: 27 and 28, 29 and 30, 31 and 32, and 33 and 34.

28. 16. The composition of any one of claims 1 to 15, wherein the second of the two or more targets is a gene of the FAD2 gene family of Glycine max.

29. 16. The composition of any one of claims 1 to 15, wherein a first of the two or more targets is an allele of the Glycine max FAD2-1A gene.

30. 16. The composition of any one of claims 1 to 15, wherein a first of the two or more targets is an allele of the Glycine max FAD2-1B gene.

31. 31. The composition of any one of claims 28 to 30, wherein the second of the two or more targets is an allele of the Glycine max FAD2-1A gene.

32. 31. The composition of any one of claims 28-30, wherein the second of the two or more targets is an allele of the Glycine max FAD2-1B gene.

33. The composition of any one of claims 1 to 15, wherein the conserved sequence is within SEQ ID NO: 25 or 26.

34. 34. The composition of any one of claims 1 to 33, wherein the composition comprises two second nucleic acids, each encoding a second transcription activator-like (TAL) effector nuclease monomer capable of binding to each of the second sequences, and at least one of the second sequences is SEQ ID NO: 17 or SEQ ID NO:

19.

35. 35. A method for simultaneously introducing mutations into two or more targets, the method comprising contacting a population of plant cells comprising said two or more targets with a composition according to any one of claims 1 to 34.

36. 1. A method for generating a soybean plant comprising a mutation that reduces expression of at least two of the FAD3A gene, the FAD3B gene, and the FAD3C gene, comprising: (a) contacting a population of soybean plant cells from a soybean plant having functional FAD3A, FAD3B, and FAD3C genes with a composition comprising: a first nucleic acid encoding a first transcription activator-like (TAL) effector nuclease monomer capable of binding to a conserved sequence of a first target gene and at least one second target gene; and a set of second nucleic acids, each second nucleic acid encoding a second TAL effector nuclease monomer capable of binding to a second sequence of the first target gene and the at least one second target gene and to at least one second target gene; contacting, wherein the conserved sequence is different from each of the second sequences, each of the second sequences is different from each other, and the conserved sequence is separated from each of the second sequences by a spacer sequence, the first TAL effector nuclease monomer can form a dimer with each of the second TAL effector nuclease monomers, the first target gene is the FAD3A gene, and the at least one second target gene is selected from the FAD3B gene and the FAD3C gene; (b) selecting from said population cells having reduced expression of said first target gene and said at least one second target gene; (c) regenerating the selected plant cells into soybean plants.

37. 37. The method of claim 36, wherein the first or second TAL effector nuclease monomer is capable of binding to a nucleic acid sequence from the group set forth in SEQ ID NOs: 4-19, or a functional variant thereof.

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