ZINC FINGER NUCLEASE-MEDIATED HOMOLOGOUS RECOMBINATION

• US 20100257638A1
• Filed: 08/09/2007
• Published: 10/07/2010
• Est. Priority Date: 08/11/2006
• 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: Garrison, Robbi J., Rubin-Wilson, Beth C., Schulenberg, Lisa L., Cai, Qihua, Worden, Andrew F., Miller, Jeffrey C., Petolino, Joseph F., Ainley, William M.
• Granted Patent: US 9,428,756 B2
• Time in Patent Office: Days
• US Class Current: 800/298
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are methods and compositions for targeted integration of an exogenous sequence into a predetermined target site in a plant genome.

Claims

1-16. -16. (canceled)

17. A method for introducing an exogenous sequence into the genome of a plant cell, the method comprising the steps of:

(a) contacting the cell with a donor DNA vector comprising first, second, and fifth DNA sequences;

wherein the first sequence is homologous to a third sequence in the genome of the plant cell, the second sequence is homologous to a fourth sequence in the genome of the plant cell, and the fifth sequence comprises an exogenous sequence and is interposed between the first and second sequences; and

(b) expressing one or more nucleases in the cell, wherein the one or more nucleases cleave chromosomal DNA at one or more corresponding target sequences between of the third and fourth sequences;

such that cleavage of chromosomal DNA in step (b) stimulates incorporation of the fifth DNA sequence into the genome by homologous recombination.

18. The method of claim 17, wherein the one or more nucleases are fusions between the cleavage domain of a Type IIS restriction endonuclease and an engineered zinc finger binding domain.

21. A transgenic plant cell obtained according to the method of claim 17.

22. A plant, or progeny thereof, comprising a transgenic plant cell according to claim 21.

61. The method of claim 17, wherein the one or more nucleases cleave chromosomal DNA at a location from 0.1 to 3 kilobase pairs from either of the first or second chromosomal sequences.

62. The method of claim 17, wherein the fifth sequence comprises sequences encoding a selectable marker.

63. The method of claim 62, wherein the selectable marker is selected from the group consisting of green fluorescent protein (GFP), β

-glucuronidase (GUS), phosphinothricin N-acetyl transferase (PAT, BAR), neomycin phosphotransferase, β

-lactamase, catechol dioxygenase, α

-amylase, tyrosinase, β

-galactosidase, luciferase, aequorin, EPSP synthase, nitrilase, acetolactate synthase (ALS), dihydrofolate reductase (DHFR), dalapon dehalogenase and anthranilate synthase.

64. The method of claim 17, wherein the fifth sequence comprises one or more transcriptional regulatory sequences.

65. The method of claim 17, wherein the fifth sequence comprises sequences encoding a protein or a portion of a protein.

66. The method of claim 65, wherein the sequences encoding the protein or the portion of the protein comprise sequences homologous to chromosomal sequences.

67. The method of claim 17, wherein the fifth sequence comprises a wild-type counterpart of a mutant chromosomal sequence, or a mutant counterpart of a wild-type chromosomal sequence.

68. The method of claim 17, wherein the first sequence has at least 35% homology to the third sequence, the second sequence has at least 35% homology to the fourth sequence, or the first sequence has at least 35% homology to the third sequence and the second sequence has at least 35% homology to the fourth sequence.

69. A seed produced from the plant, or progeny thereof, of claim 22.

19. (canceled)

20. (canceled)

23. A method for intramolecular homologous recombination in the genome of a plant cell, the method comprising the steps of:

(a) contacting a DNA segment within the genome of the plant cell, the DNA segment comprising a first sequence, a second sequence homologous to the first sequence, and a third sequence interposed between the first and second sequences,

with a zinc finger nuclease, wherein the nuclease cleaves the DNA segment within the third sequence;

wherein DNA between the first and second sequences is deleted from the chromosome.

29. The method of claim 23, wherein the nuclease is a pair of fusion proteins, wherein each fusion protein is a fusion between the cleavage domain of a Type IIS restriction endonuclease and an engineered zinc finger binding domain.

30. The method of claim 23, wherein the third sequence is at least 100 base pairs from the first sequence, at least 100 base pairs from the second sequence, or at least 100 base pairs from the first sequence and the second sequence.

33. The method of claim 23, wherein at least one of the first or second sequences is exogenous to the organism.

37. The method of claim 23, wherein the deleted DNA is replaced by an exogenous sequence, the method further comprising:

introducing a polynucleotide into the cell, wherein the polynucleotide comprises;

(a) fourth and fifth sequences, wherein the fourth sequence is homologous to non-deleted sequences in proximity to the first sequence and the fifth sequence is homologous to non-deleted sequences in proximity to the second sequence; and

(b) the exogenous sequence.

38. The method of claim 37 wherein the exogenous sequence is a selectable marker.

39. The method of claim 38, wherein the selectable marker is selected from the group consisting of green fluorescent protein (GFP), β

-glucuronidase (GUS), phosphinothricin N-acetyl transferase (PAT, BAR), neomycin phosphotransferase, β

-lactamase, catechol dioxygenase, α

-amylase, tyrosinase, β

-galactosidase, luciferase, aequorin, EPSP synthase, nitrilase, acetolactate synthase (ALS), dihydrofolate reductase (DHFR), dalapon dehalogenase and anthranilate synthase.

70. A transgenic plant cell obtained according to the method of claim 37.

71. A plant, or progeny thereof, comprising a transgenic plant cell according to claim 70.

72. A seed produced from the plant, or progeny thereof, of claim 71.

24-28. -28. (canceled)

31. (canceled)

32. (canceled)

34. (canceled)

40-60. -60. (canceled)