FAD2 PERFORMANCE LOCI AND CORRESPONDING TARGET SITE SPECIFIC BINDING PROTEINS CAPABLE OF INDUCING TARGETED BREAKS

• US 20140090112A1
• Filed: 09/05/2013
• Published: 03/27/2014
• Est. Priority Date: 09/07/2012
• Status: Active Grant

• Current Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Therapeutics, Inc.
• Original Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Biosciences, Inc.
• Inventors: Spangenberg, German, Webb, Steven R., Gupta, Manju, Ainley, William Michael, Henry, Matthew J., Miller, Jeffrey C., Cogan, Noel, Forster, John, Hayden, Matthew, Sawbridge, Tim, Guschin, Dmitry Y.
• Granted Patent: US 9,963,711 B2
• Time in Patent Office: Days
• US Class Current: 800/298
• 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

A method of gene editing or gene stacking within a FAD2 loci by cleaving, in a site directed manner, a location in a FAD2 gene in a cell, to generate a break in the FAD2 gene and then ligating into the break a nucleic acid molecule associated with one or more traits of interest is disclosed.

Claims

1. A method for integrating a nucleic acid sequence of interest into a specific site in the genome of a cell, the method comprising:

cleaving, in a site specific matter, a target site in a FAD2 gene in a cell, to thereby generate a break in the FAD2 gene;

integrating into the break a nucleic acid of interest.

2. The method according to claim 1, wherein the FAD2 gene is a FAD2A, FAD2A′

, FAD2C and/or a FAD2C′

gene.

3. The method according to claim 1, wherein the cleaving in a site specific manner comprises introducing a fusion protein comprising a DNA-binding domain and a cleavage domain or cleavage half-domain into the cell, wherein the fusion protein binds with specificity to the target site and cleaves at or near the target site to thereby generate the break.

4. The method according to claim 3, wherein the DNA-binding domain is selected from a group comprising a meganuclease DNA-binding domain, a leucine zipper DNA-binding domain, a transcription activator-like (TAL) DNA-binding domain, a recombinase, a RNA-guided CRISPR-Cas9, a zinc finger protein DNA-binding domain, and chimeric combinations of any of the foregoing.

5. The method according to claim 3, 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.

6. The method according to claim 3, wherein the zinc finger protein is a zinc finger nuclease.

7. The method according to claim 6, wherein the zinc finger nuclease 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 4.

8. The method according to claim 2, wherein the cleaving in a site specific manner is specific for some but not all copies of FAD2A, FAD2A′

, FAD2C and/or FAD2C′

.

9. The method according to claim 1, wherein the cleaving in a site specific manner comprises cleaving at site selected from the group consisting of SEQ ID NOs:

22-26, and SEQ ID NOs;

28-33 and SEQ ID NOs;

35-38.

10. The method according to claim 1, wherein integrating into the break the nucleic acid sequence of interest does not have an adverse impact on the agronomic or quality properties of the cell.

11. The method according to claim 1, wherein the cell is a plant, fungi, bacteria or algae cell.

12. The method according to claim 11, wherein the plant cell is a monocot plant cell or a dicot plant cell.

13. The method according to claim 12, 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.

14. The method according to claim 1, wherein the break is a double strand break.

15. The method according to claim 1, wherein the nucleic acid of interest is heterologous to the cell.

16. The method according to claim 1, wherein the nucleic acid sequence of interest encodes a protein.

17. The method according to claim 16, wherein the nucleic acid sequence of interest is selected from the group consisting of insecticidal resistance genes, herbicide tolerance genes, nitrogen use efficiency genes, water use efficiency genes, nutritional quality genes, DNA binding genes, and selectable marker genes.

18. The method according to claim 1, wherein the nucleic acid sequence of interest comprises a DNA-binding domain binding site.

19. The method according to claim 3, wherein the fusion protein is introduced into the cell as a polynucleotide encoding the fusion protein.

20. A transgenic cell comprising a nucleotide sequence integrated into a FAD2A, FAD2A′

, FAD2C and/or FAD2C′

gene.

21. The transgenic cell of claim 20, wherein the nucleotide sequence is heterologous to the cell.

22. The transgenic cell of claim 20, wherein the nucleotide sequence is homologous to the cell.

23. The transgenic cell of claim 22, wherein the homologous sequence comprises at least one single nucleotide polymorphism.

24. The transgenic cell of claim 20, wherein the nucleotide sequence is integrated into some but not all copies of the FAD2A, FAD2A′

, FAD2C and/or FAD2C′

gene.

25. The transgenic cell of claim 20, wherein the nucleic acid sequence is integrated at a target site selected from the group consisting of SEQ ID NOs:

22-26, SEQ ID NOs;

28-33 and SEQ ID NOs;

35-38.

26. A transgenic plant or seed, comprising the transgenic cell of claim 20.

27. The transgenic cell of claim 20, wherein presence of the nucleotide sequence in the FAD2A, FAD2A′

, FAD2C and/or FAD2C′

gene does not negatively influence the agronomic or quality properties of the cell.

28. A site specific zinc finger nuclease that cleaves at or near a nucleic acid target site selected from the group consisting of SEQ ID NOs:

22-26 and SEQ ID NOs;

28-33 and SEQ ID NOs;

35-38.

29. The zinc finger nuclease of claim 28, wherein the zinc finger nuclease 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 4.

30. A method of cleaving a FAD2 loci in a plant cell, the method comprising:

introducing a fusion protein comprising a DNA-binding domain and a cleavage domain or cleavage half-domain into the cell, wherein the fusion protein binds with specificity to a target site in the FAD2 loci and cleaves at or near the target site to thereby generate a break.

31. The method according to claim 30, further comprising integrating into the break a nucleic acid molecule of interest.

32. The method according to claim 30, wherein the FAD2 loci is a FAD2A, FAD2A′

, FAD2C and/or FAD2C′

gene.

33. The method according to claim 30, 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.

34. The method according to claim 30, wherein the cleavage domain or cleavage half-domain is selected from the 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.

35. The method according to claim 30, wherein the fusion protein is a zinc finger nuclease.

36. The method according to claim 30, wherein the zinc finger nuclease comprises five or six zinc finger domains ordered finger 1 to finger 5 or finger 1 to finger 6, 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 4.

37. The method according to claim 30, wherein the cleaving is specific for some but not all copies FAD2A, FAD2A′

, FAD2C and/or FAD2C′

.

38. The method according to claim 30, wherein the target site is selected from the group consisting of SEQ ID NOs:

22-26, SEQ ID NOs;

28-33 and SEQ ID NOs;

35-38.