Tal effector nucleases for gene editing

Abstract

Soybean plants, plant parts and plant cells capable of producing seeds comprising oil having relatively higher oleic acid levels and lower linoleic and linolenic acid levels than corresponding seeds lacking the targeted mutation.SOLUTION: Disclosed are TALEN compositions and methods of use, including using a multiplexing composition to make targeted mutations in several genes at once, such as the FAD3A / B / C genes, compositions for making targeted mutations in a single gene, such as a gene encoding a FAD2 protein, and combinations thereof. The compositions and methods can provide genetically edited plants, plant parts, and plant cells with improved characteristics as compared to corresponding unmodified plants, plant parts, or plant cells.SELECTED DRAWING: FIG. 1A

Claims

1. A soybean plant, a soybean plant part, or a soybean plant cell comprising one or more mutations that reduce the expression of at least one of the FAD2-1A gene and the FAD2-1B gene, Compared to oil produced from a corresponding soybean plant, soybean plant part, or soybean plant cell lacking one or more of the aforementioned mutations, it produces an oil having an increased oleic acid content. At least one mutation is induced by a low-frequency cleavage endonuclease that can bind to the nucleic acid sequence to introduce one or more mutations into the target sequences of the FAD2-1A and FAD2-1B genes. The nucleic acid sequence is selected from the group of sequence numbers 27-34 or their functional variants, The target sequence includes sequence numbers 20, 21, 22, 25, or 26 or their functional variants. Soybean plant, soybean plant part, or soybean plant cell.

2. A soybean plant, a portion of a soybean plant, or a soybean plant cell according to claim 1, selected from the group consisting of cotyledon cells, seeds, embryos, embryogenic callus cells, and pollen cells.

3. The soybean plant, soybean plant portion, or soybean plant cell according to claim 1 or 2, wherein the low-frequency cleavage endonuclease is a transcription activator-like (TAL) effector nuclease, a meganuclease, a zinc finger nuclease, or CRISPR / Cas9.

4. The soybean plant, soybean plant portion, or soybean plant cell according to claim 3, wherein the TAL effector nuclease comprises a pair of monomers bound to a sequence selected from the following group. Sequence IDs 27 and 28; Sequence IDs 29 and 30; Sequence IDs 31 and 32; and Sequence IDs 33 and 34.

5. A soybean plant, a portion of a soybean plant, or a soybean plant cell according to claim 1, which does not contain a transgene.

6. The soybean plant, soybean plant part, or soybean plant cell according to claim 1, which produces an oil having one or more of the following characteristics: increased oleic acid content, decreased linoleic acid content, and decreased linolenic acid content, compared to the oil produced from a corresponding soybean plant, soybean plant part, or soybean plant cell lacking one or more of the aforementioned mutations.

7. The second mutation further includes reducing the expression of at least two of the FAD3A, FAD3B, and FAD3C genes, The soybean plant, soybean plant part, or soybean plant cell according to claim 1, which produces an oil having a reduced linolenic acid content compared to the oil produced from a corresponding soybean plant, soybean plant part, or soybean plant cell lacking the second mutation.

8. The soybean plant, soybean plant part, or soybean plant cell according to claim 7, which produces an oil having an oleic acid content of at least 79% by weight, a linoleic acid content of less than 6.5% by weight, and a linolenic acid content of less than 3.2% by weight.

9. Soybean plant, soybean plant part, or soybean plant cell, 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 A first set of mutations comprising one or more FAD3A alleles, one or more FAD3B alleles, and one or more FAD3C alleles, The first set of mutations is A first nucleic acid encoding a first transcription activator-like (TAL) effector nuclease monomer that can bind to the first half-site sequence of the first target gene, A second set of nucleic acids, each of which encodes a second TAL effector nuclease monomer capable of binding to the second half-site sequence of the first target gene and at least one second target gene, is induced by the expression of the second set of nucleic acids, The first half-site sequence is a conserved sequence, The first half-site array and each second half-site array are different and separated by a spacer array. A first set of mutations such that the first TAL effector nuclease monomer can form dimers with each of the second TAL effector nuclease monomers, One or more FAD2-1A alleles, One or more FAD2-1B alleles, or This includes mutations in one or more FAD2-1A alleles and one or more FAD2-1B alleles, Compared to oil produced from a corresponding soybean plant, soybean plant part, or soybean plant cell lacking the aforementioned mutation, it produces an oil having a reduced linolenic acid content, an increased oleic acid content, and a reduced linoleic acid content. Soybean plant, plant part, or plant cell.

10. The soybean plant, soybean plant portion, or soybean plant cell according to claim 9, wherein the TAL effector nuclease comprises a pair of monomers bound to a sequence selected from the following group. Sequence IDs 27 and 28; Sequence IDs 29 and 30; Sequence IDs 31 and 32; and Sequence IDs 33 and 34.

11. A soybean plant, a portion of a soybean plant, or a soybean plant cell according to claim 9, which does not contain a transgene.

12. A method for producing soybean plants containing a mutation that reduces the expression of at least one of the FAD2-1A gene and the FAD2-1B gene, A population of soybean plant cells from soybean plants possessing functional FAD2-1A and FAD2-1B genes is brought into contact with one or more nucleic acid sequences encoding low-frequency cleavage endonucleases capable of binding to the nucleic acid sequence to introduce one or more mutations into the target sequences of the FAD2-1A and FAD2-1B genes, wherein the nucleic acid sequence is selected from the group of SEQ ID NOs. 27-34 or their functional variants, and the target sequence includes SEQ ID NOs. 20, 21, 22, 25, or 26 or their functional variants. From the aforementioned population, select soybean plant cells in which the expression of the FAD2-1A gene or the FAD2-1B gene is reduced by a low-frequency cleavage endonuclease, A method comprising regenerating the selected soybean plant cells into a soybean plant.

13. The method according to claim 12, wherein the low-frequency cleavage endonuclease is a transcription activator-like (TAL) effector nuclease, a meganuclease, a zinc finger nuclease, or CRISPR / Cas9.

14. The method according to claim 13, wherein the TAL effector nuclease comprises a pair of monomers that bind to a nucleic acid sequence selected from the following group. Sequence IDs 27 and 28; Sequence IDs 29 and 30; Sequence IDs 31 and 32; and Sequence IDs 33 and 34.

15. The method according to claim 12, wherein the soybean plant does not contain introduced genes.

16. The method according to claim 12, wherein a soybean plant produces an oil having one or more of the following characteristics compared to oil produced from a corresponding soybean plant lacking one or more of the aforementioned mutations: an increase in oleic acid content, a decrease in linoleic acid content, and a decrease in linolenic acid content.

17. The second mutation further includes reducing the expression of at least two of the FAD3A, FAD3B, and FAD3C genes, Soybean plants produce oil having a reduced linolenic acid content compared to oil produced from corresponding soybean plants lacking the second mutation, optionally, The method according to claim 12, wherein a soybean plant produces an oil having an oleic acid content of at least 79% by weight, a linoleic acid content of less than 6.5% by weight, and a linolenic acid content of less than 3.2% by weight.

18. A soybean plant produced by a method comprising mutations that reduce the expression of at least two of the FAD3A, FAD3B, and FAD3C genes, The method is, Contacting a population of soybean plant cells from soybean plants possessing functional FAD3A, FAD3B, and FAD3C genes with the composition, where, The composition is A first nucleic acid encoding a conserved sequence of a first target gene and a first transcription activator-like (TAL) effector nuclease monomer capable of binding to at least one second target gene, A set of second nucleic acids, each of which encodes 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, The stored sequence is different from each of the second sequences, and each of the second sequences is different from one another. The stored array is separated from each of the second arrays by a spacer array. The first TAL effector nuclease monomer can form dimers with each of the second TAL effector nuclease monomers. The first target gene is the FAD3A gene, The at least one second target gene is selected from the FAD3B gene and the FAD3C gene, From the aforementioned population, select soybean plant cells in which the expression of the first target gene and the at least one second target gene is reduced, and This includes regenerating the selected soybean plant cells into soybean plants. Soybean plant.

19. A soybean oil composition comprising soybean oil produced by the soybean plant described in claim 18, Soybean oil has one or more of the following characteristics compared to oil produced from a corresponding soybean plant, plant part, or plant cell lacking the mutations in at least two genes: increased oleic acid content, decreased linoleic acid content, and decreased linolenic acid content. Soybean oil composition.

20. A soybean oil composition comprising soybean plants, soybean plant parts, or soybean oil produced by soybean plant cells, containing one or more mutations that reduce the expression of at least one of the FAD2-1A gene and FAD2-1B gene, Soybean oil has one or more of the following characteristics compared to oil produced from a corresponding soybean plant, soybean plant part, or soybean plant cell lacking one or more of the aforementioned mutations: an increase in oleic acid content, a decrease in linoleic acid content, and a decrease in linolenic acid content. The at least one mutation comprises a target mutation in a target sequence that is induced by a low-frequency cleavage endonuclease capable of binding to a nucleic acid sequence selected from the group of SEQ ID NOs. 27-34 or its functional variants, The target sequence includes sequence numbers 20, 21, 22, 25, or 26 or their functional variants. Soybean oil composition.

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