Fad2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks

• US 10,287,595 B2
• Filed: 10/13/2016
• Issued: 05/14/2019
• Est. Priority Date: 09/07/2012
• Status: Active Grant

• Current Assignee: Dow Agrosciences LLC (Corteva, Inc.)
• Original Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Therapeutics, Inc.
• Inventors: Ainley, W. Michael, Webb, Steven R., Samuel, Jayakumar P., Guschin, Dmitry Y., Miller, Jeffrey C., Zhang, Lei
• Primary Examiner(s): Keogh, Matthew R
• Application Number:
  US15/292,774
• Publication Number:
  US 20170044559A1
• Time in Patent Office: 943 Days
• Field of Search: None
• CPC Class Codes: A01H 5/10 Seeds
• CPC Class Codes: A01H 6/542 Glycine max [soybean]
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

Methods and compositions for gene disruption, gene editing or gene stacking within a FAD2 loci by cleaving, in a site directed manner, a location in a FAD2 gene in a soybean cell, to generate a break in the FAD2 gene and then optionally integrating into the break a nucleic acid molecule of interest is disclosed.

Claims

1. A zinc finger protein that binds to a nucleic acid target site comprising any of SEQ ID NO:

14 to SEQ ID NO;

20.

2. The zinc finger protein of claim 1, wherein the zinc finger protein comprises from three to six zinc finger domains, each zinc finger domain comprising a recognition helix region, wherein the zinc finger protein comprises the recognition helix regions ordered and shown in a single row of Table 2.

3. A zinc finger nuclease comprising a zinc finger protein of claim 1 and a cleavage domain.

4. A polynucleotide comprising a sequence encoding a zinc finger protein according to claim 1.

5. A polynucleotide comprising a sequence encoding a zinc finger nuclease according to claim 3.

6. An isolated cell comprising a zinc finger protein according to claim 1.

7. An isolated cell comprising a polynucleotide according to claim 4.

8. A method of cleaving a FAD2 loci in a soybean cell, the method comprising:

introducing a polynucleotide encoding the zinc finger nuclease (ZFN) according to claim 3 into the cell, wherein the ZFN cleaves the FAD2 loci and further wherein the ZFN comprises a pair of zinc finger proteins as shown in Table 2 as follows;

a pair comprising the ZFPs designated 37354 and 37355;

a pair comprising the ZFPs designated 37370 and 37371;

a pair comprising the ZFPs designated 37374 and 37375;

a pair comprising the ZFPs designated 37366 and 37367;

a pair comprising the ZFPs designated 37384 and 37385;

a pair comprising the ZFPs designated 37398 and 37399;

or a pair comprising the ZFPs designated 37392 and 37393.

9. The method according to claim 8, further comprising integrating a nucleic acid molecule of interest into the cleaved FAD2 loci.

10. The method according to claim 9, wherein the FAD2 loci is a FAD2 2.3 gene, FAD2 2.6 gene, or both.

11. The method according to claim 10, wherein the cleavage domain is selected from the group consisting of a cleavage half-domain from a type IIS restriction endonuclease, a cleavage half-domain from Fold endonuclease, a cleavage half-domain from StsI endonuclease, and a homing endonuclease.