FAD3 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks

• US 10,526,610 B2
• Filed: 01/29/2018
• Issued: 01/07/2020
• Est. Priority Date: 09/07/2012
• Status: Active Grant

• Current Assignee: Dow Agrosciences LLC (Corteva, Inc.)
• Original Assignee: Dow Agrosciences LLC (Corteva, Inc.)
• Inventors: Cogan, Noel, Forster, John, Hayden, Matthew, Sawbridge, Tim, Spangenberg, German, Webb, Steven R., Gupta, Manju, Ainley, W. Michael, Henry, Matthew J., Miller, Jeffrey C., Guschin, Dmitry Y.
• Primary Examiner(s): Keogh, Matthew R
• Application Number:
  US15/882,609
• Publication Number:
  US 20180163217A1
• Time in Patent Office: 708 Days
• Field of Search: None
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/8241 Phenotypically and genetically modified plants via recombinant DNA technology
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

A method of gene editing or gene stacking within a FAD3 loci by cleaving, in a site directed manner, a location in a FAD3 gene in a cell, to generate a break in the FAD3 gene and then ligating into the break a nucleic acid molecule associated with one or more traits of interest is disclosed.

Claims

1. A nuclease comprising a cleavage domain and a DNA-binding domain that binds to a target site as shown in any of SEQ ID NOs:

20-23, SEQ ID NOs;

25-38, SEQ ID NOs;

40-45, SEQ ID NO;

47 or SEQ ID NO;

49, wherein the nuclease cleaves a FAD3 gene in a plant cell.

2. The nuclease of claim 1, wherein the DNA-binding domain is selected from the group consisting of a meganuclease DNA-binding domain, a leucine zipper DNA-binding domain, a transcription activator-like (TAL) DNA-binding domain, a RNA-guided CRISPR-Cas9, a recombinase, a zinc finger protein DNA-binding domain, and chimeric combinations of any of the foregoing.

3. The nuclease of claim 2, wherein the zinc finger protein DNA-binding domain 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 3.

4. The nuclease according to claim 1, wherein the cleavage domain or cleavage half-domain is selected from a group consisting of a cleavage half-domain from a type IIS restriction endonuclease, a cleavage half-domain from FokI endonuclease, a cleavage half-domain from StsI endonuclease, and a homing endonuclease.

5. The nuclease of claim 1, wherein the nuclease binds to some but not all copies of a FAD3A, FAD3A′

, FAD3A″

, FAD3C, FAD3C″ and

/or a FAD3C′

gene.

6. The nuclease according to claim 1, wherein the plant cell is a monocot plant cell or a dicot plant cell.

7. The nuclease according to claim 6, wherein the plant cell is selected from the group consisting of Brassica sp., Brassica napus;

Brassica rapa;

Brassica juencea;

Brassica oleracea;

Brassica nigra;

Zea sp.;

Zea mays;

Glycine sp.;

Glycine max;

Triticum sp;

Triticum aestivum;

Oryza sp;

Oryza sativa;

Triticae sp.;

Triticae triticum;

Heliantheae sp.;

Heliantheae helianthus;

Gossypium sp.;

Gossypium hirsutum; and

Hordeum vulgar.

8. A method of cleaving a FAD3 loci in a plant cell, the method comprising:

introducing a polynucleotide comprising a nuclease according to claim 1 into the cell such that the FAD3 gene in the plant cell is cleaved.

9. The method according to claim 8, further comprising integrating a nucleic acid molecule of interest into the cleaved FAD3 gene.

10. The method according to claim 8, wherein the FAD3 loci is a FAD3A, FAD3A′

, FAD3A″

, FAD3C, FAD3C″ and

/or a FAD3C′

gene.