METHODS AND COMPOSITIONS FOR INTEGRATION OF AN EXOGENOUS SEQUENCE WITHIN THE GENOME OF PLANTS

• US 20140304853A1
• Filed: 04/02/2014
• Published: 10/09/2014
• Est. Priority Date: 04/05/2013
• Status: Active Grant

• Current Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Therapeutics, Inc.
• Original Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Guschin, Dmitry Y., Hayden, Matthew, Mason, John, Miller, Jeffrey C., Petolino, Joseph F., Sawbridge, Tim, Spangenberg, German, Webb, Steven R., Ainley, W. Michael, Isenegger, Daniel, Ran, Yidong
• Granted Patent: US 10,501,748 B2
• Time in Patent Office: Days
• US Class Current: 800/278
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8274 for herbicide resistance
• CPC Class Codes: C12N 15/8278 Sulfonylurea
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

Disclosed herein are methods and compositions for parallel or sequential transgene stacking in plants to produce plants with selected phenotypes.

Claims

1. A plant cell comprising a targeted genomic modification to one or more alleles of an endogenous gene in the plant cell, wherein the genomic modification follows cleavage by a site specific nuclease, and wherein the genomic modification produces a mutation in the endogenous gene such that the endogenous gene produces a product that results in an herbicide tolerant plant cell.

2. The plant cell of claim 1, wherein the genomic modification comprises integration of one or more exogenous sequences.

3. The plant cell of claim 1, wherein the genomic modification comprises introduction of one or more indels that disrupt expression of the endogenous gene.

4. The plant cell of claim 1, wherein the endogenous gene with the genomic modification encodes a protein that confers tolerance to sulfonylurea herbicides.

5. The plant cell of claim 1, wherein the endogenous gene with the genomic modification encodes a protein that confers tolerance to imidazolinone herbicides.

6. The plant cell of claim 2, wherein the exogenous sequence does not encode a transgenic selectable marker.

7. The plant cell of claim 2, wherein the exogenous sequence encodes a protein selected from the group consisting of a protein that increases crop yield, a protein encoding disease resistance, a protein that increases growth, a protein encoding insect resistance, a protein encoding herbicide tolerance, and combinations thereof.

8. The plant cell of claim 7, wherein the increased crop yield comprises an increase in fruit yield, grain yield, biomass, fruit flesh content, size, dry weight, solids content, weight, color intensity, color uniformity, altered chemical characteristics, or combinations thereof.

9. The plant cell of claim 1, wherein the endogenous gene is an endogenous acetohydroxyacid synthase (AHAS) gene.

10. The plant cell of claim 2, wherein two or more exogenous sequences are integrated into the endogenous gene.

11. The plant cell of claim 1, wherein the plant cell is a polyploid plant cell.

12. The plant cell of claim 1, wherein the site specific nuclease comprises a zinc finger DNA-binding domain, and a FokI cleavage domain.

13. The plant cell of claim 12, wherein the zinc finger DNA-binding domain encodes a protein that binds to a target site selected from the group consisting of SEQ ID NOs:

35-56 and 263-278.

14. The plant cell of claim 1, wherein the plant is selected from the group consisting of wheat, soy, maize, potato, alfalfa, rice, barley, sunflower, tomato, Arabidopsis, cotton, Brassica species, and timothy grass.

15. A plant, plant part, seed, or fruit comprising one or more plant cells of claim 1.

16. The plant cell of claim 1, further comprising one or more transgenes integrated into the genome of the plant cell at one or more loci different from the endogenous gene.

17. A method for making a plant cell according to claim 1, the method comprising:

expressing one or more site specific nucleases in the plant cell; and

modifying one or more alleles of an endogenous gene across multiple genomes of a polyploid plant cell.

18. The method of claim 17, wherein the endogenous gene is an acetohydroxyacid synthase (AHAS) gene.

19. The method of claim 17, wherein the modification disrupts expression of the endogenous gene.

20. The method of claim 17, wherein the modification comprises integration of one or more exogenous sequences into one or more alleles of the endogenous gene.

21. A plant, plant part, seed, or fruit comprising one or more plant cells produced by the method of claim 17.

22. A zinc finger protein that binds to a target site selected from the group consisting of SEQ ID NOs:

35-56 and 263-278.

23. The zinc finger protein of claim 22, comprising the recognition helix regions shown in a single row of Table 2 or Table 12.

24. A method of integrating one or more exogenous sequences into the genome of a plant cell, the method comprising:

a) expressing one or more site specific nucleases in the plant cell, wherein the one or more nucleases target and cleave chromosomal DNA of one or more endogenous loci;

b) integrating one or more exogenous sequences into the one or more endogenous loci within the genome of the plant cell, wherein the one or more endogenous loci are modified such that the endogenous gene is mutated to express a product that results in a selectable phenotype in the plant cell; and

c) selecting plant cells that express the selectable phenotype, wherein plant cells are selected which incorporate the one or more exogenous sequences.

25. The method of claim 24, wherein the one or more exogenous sequences are selected from the group consisting of a donor polynucleotide, a transgene, or any combination thereof.

26. The method of claim 24, wherein integrating the one or more exogenous sequences occurs by homologous recombination or non-homologous end joining.

27. The method of claim 24, wherein the one or more exogenous sequences are incorporated simultaneously or sequentially into the one or more endogenous loci.

28. The method of claim 24, wherein the one or more endogenous loci comprise an acetohydroxyacid synthase (AHAS) gene.

29. The method of claim 28, wherein the AHAS gene is located on an A, B, or D genome of a polyploidy genome.

30. The method of claim 28, wherein the one or more exogenous sequences are integrated into the AHAS gene.

31. The method of claim 30, wherein the one or more exogenous sequences encode a S653N AHAS mutation.

32. The method of claim 30, wherein the one or more exogenous sequences encode a P197S AHAS mutation.

33. The method of any of claim 24, wherein the site specific nuclease is selected from the group consisting of a zinc finger nuclease, a TAL effector domain nuclease, a homing endonuclease, and a Crispr/Cas single guide RNA nuclease.

34. The method of claim 33, wherein the site specific nuclease comprises a zinc finger DNA-binding domain and a FokI cleavage domain.

35. The method of any claim 24, wherein the one or more exogenous sequences encode a transgene or produce an RNA molecule.

36. The method of claim 35, wherein the transgene encodes a protein selected from the group consisting of a protein that increases crop yield, a protein encoding disease resistance, a protein that increases growth, a protein encoding insect resistance, a protein encoding herbicide tolerance, and combinations thereof.

37. The method of claim 35, wherein the integration of the transgene further comprises introduction of one or more indels that disrupt expression of the one or more endogenous loci and produce the selectable phenotype.

38. The method of claim 24, the method further comprising the steps of;

d) culturing the selected plant cells comprising the one or more exogenous sequences; and

e) obtaining a whole plant comprising the one or more exogenous sequences integrated within the one or more endogenous loci of the plant genome.

39. The method of claim 24, wherein a selection agent comprising an imidazolinone, or a sulfonylurea selection agent is used to select the plant cells.

40. The method of claim 38, wherein the whole plant comprising the one or more exogenous sequences integrated within the one or more endogenous loci of the plant genome is further modified to incorporate an additional exogenous sequence within the endogenous loci of the plant genome.

41. The method of claim 38, wherein the one or more exogenous sequence does not encode a transgenic selectable marker.