Engineered cleavage half-domains

• US 20090311787A1
• Filed: 07/02/2008
• Published: 12/17/2009
• Est. Priority Date: 05/25/2006
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Miller, Jeffrey C.
• Granted Patent: US 7,914,796 B2
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are engineered cleavage half-domains; fusion polypeptides comprising these engineered cleavage half-domains; polynucleotides encoding the engineered cleavage half-domains and fusion proteins; and cells comprising said polynucleotides and/or fusion proteins. Also described are methods of using these polypeptides and polynucleotides, for example for targeted cleavage of a genomic sequence.

Claims

1. A method for cleaving DNA in a cell, the method comprising:

expressing first, second, third and fourth fusion proteins in the cell, each of the fusion proteins comprising;

(i) a zinc finger DNA-binding domain that binds to a target site in the DNA, and

(ii) an engineered FokI cleavage half-domain;

further wherein;

(a) the first fusion protein comprises the engineered FokI cleavage half-domain designated (i) Q486E or (ii) E490K;

(b) the second fusion protein comprises the engineered FokI cleavage half-domain designated (i) I499L if the first fusion protein comprises Q486E or (ii) I538K if the first fusion protein comprises E490K;

(c) the third fusion protein comprises the engineered FokI cleavage half-domain designated R487D;

(c) the fourth fusion protein comprises the engineered FokI cleavage half-domain designated D483R;

(d) the first and second fusion proteins bind to first and second target sites respectively, wherein a first cleavage site lies between the first and second target sites, and

(c) the third and fourth fusion proteins bind to third and fourth target sites respectively, wherein a second cleavage site lies between the third and fourth target sites;

such that the first and second fusion proteins cleave the DNA at the first cleavage site, the third and fourth fusion proteins cleave the DNA at the second cleavage site.

2. The method of claim 1, wherein the DNA is in cellular chromatin.

3. The method of deleting sequences in cellular DNA, the method comprising, cleaving cellular DNA according to the method of claim 1, the DNA ends are rejoined such that sequences between the first and second cleavage sites are deleted.

4. The method of claim 3, wherein the first and second cleavage sites are on the same chromosome.

5. The method of claim 1, wherein the cell is a eukaryotic cell.

6. The method of claim 5, wherein the cell is a mammalian cell.

7. The method of claim 6, wherein the cell is a human cell.

8. The method of claim 5, wherein the cell is a plant cell.

9. A method for targeted replacement of a genomic sequence, the method comprising:

(a) cleaving a genomic DNA sequence according to the method of claim 1; and

(b) contacting the cell with a donor polynucleotide, wherein the donor polynucleotide comprises.;

(i) sequences homologous to genomic sequences flanking the first and second cleavage sites; and

(ii) an exogenous polynucleotide;

whereby genomic sequences between the first and second cleavage sites are replaced by the exogenous polynucleotide.

10. The method of claim 9, wherein the exogenous polynucleotide comprises a sequence that is homologous but non-identical sequence to the genomic sequence between the first and second cleavage sites.

11. The method of claim 10, wherein the homologous but non-identical sequence comprises a deletion with respect to the genomic sequences.

12. The method of claim 10, wherein the homologous but non-identical sequence comprises an insertion with respect to the genomic sequences.

13. The method of claim 9, wherein the exogenous polynucleotide comprises a sequence that is non-homologous to genomic sequences between the first and second cleavage sites.

14. The method of claim 9, wherein the cell is arrested in the G2 phase of the cell cycle.

15. The method of claim 9, wherein the cell is a eukaryotic cell.

16. The method of claim 15, wherein the cell is a mammalian cell.

17. The method of claim 16, wherein the cell is a human cell.

18. The method of claim 15, wherein the cell is a plant cell.