Targeted deletion of cellular DNA sequences

• US 8,409,861 B2
• Filed: 12/15/2005
• Issued: 04/02/2013
• Est. Priority Date: 08/08/2003
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Guschin, Dmitry, Holmes, Michael C.
• Primary Examiner(s): DUNSTON, JENNIFER ANN
• Application Number:
  US11/304,981
• Publication Number:
  US 20060188987A1
• Time in Patent Office: 2,665 Days
• Field of Search: None
• US Class Current: 435/463
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are methods and compositions for targeted deletion of double-stranded DNA. The compositions include fusion proteins comprising a cleavage domain (or cleavage half-domain) and an engineered zinc finger domain, and polynucleotides encoding same. Methods for targeted deletion include introduction of such fusion proteins, or polynucleotides encoding same, into a cell such that two targeted cleavage events occur. Subsequent cellular repair mechanisms result in deletion of sequences between the two cleavage sites.

Claims

1. A method for deleting sequences in a region of interest in double-stranded DNA of genomic cellular chromatin 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) a FokI cleavage half-domain,

wherein the cleavage half-domain of at least one of the fusion proteins comprises an alteration in amino acid 490 of the wild-type dimerization interface of the cleavage half-domain; and

further wherein;

(a) 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

(b) 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, and DNA ends are rejoined such that sequences between the first and second cleavage sites are deleted.

2. The method of claim 1, wherein the first and second cleavage sites are on the same chromosome.

3. The method of claim 1, wherein the near edges of the first and second target sites are separated by between 4 and 6 nucleotide pairs.

4. The method of claim 1, wherein the near edges of the third and fourth target sites are separated by between 4 and 6 nucleotide pairs.

5. The method of claim 2, wherein the cell is an isolated human cell, and the region of interest is in a CCR5 gene.

6. The method of claim 1, wherein the cell is a primary cell.

7. The method of claim 1, wherein the first and second cleavage sites are separated by more than 100 nucleotide pairs.

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

(a) 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) a FokI cleavage half-domain;

wherein the cleavage half-domain of at least one of the fusion proteins comprises an alteration in amino acid 490 of the wild-type dimerization interface of the cleavage half-domain

wherein 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

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, and the third and fourth fusion proteins cleave the DNA at the second cleavage site; 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) sequences homologous but non-identical to genomic sequences between the first and second cleavage sites;

whereby genomic sequences between the first and second cleavage sites are replaced by the homologous but non-identical sequences of the donor polynucleotide.

9. The method of claim 8, wherein the homologous but non-identical sequences comprise a deletion with respect to the genomic sequences.

10. The method of claim 8, wherein the homologous but non-identical sequences comprise an insertion with respect to the genomic sequences.

11. The method of claim 9, wherein the homologous but non-identical sequences comprise an insertion with respect to the genomic sequences.

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

(a) 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) a FokI cleavage half-domain,

wherein the cleavage half-domain of at least one of the fusion proteins comprises an alteration in amino acid 490 of the wild-type dimerization interface of the cleavage half-domain;

wherein 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

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, and the third and fourth fusion proteins cleave the DNA at the second cleavage site; 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) sequences that are non-homologous to genomic sequences between the first and second cleavage sites;

whereby genomic sequences between the first and second cleavage sites are replaced by the non-homologous sequences of the donor polynucleotide.