Methods and compositions for targeted cleavage and recombination

• US 20070218528A1
• Filed: 02/03/2005
• Published: 09/20/2007
• Est. Priority Date: 02/05/2004
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Miller, Jeffrey
• Granted Patent: US 7,972,854 B2
• Time in Patent Office: Days
• US Class Current: 435/91.200
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• 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 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are methods and compositions for targeted cleavage of a genomic sequence, targeted alteration of a genomic sequence, and targeted recombination between a genomic region and an exogenous polynucleotide homologous to the genomic region. The compositions include fusion proteins comprising a cleavage domain (or cleavage half-domain) and an engineered zinc finger domain, as well as polynucleotides encoding same. Fusion proteins comprising cleavage half-domains are used in pairs, to reconstitute a functional cleavage domain. In these fusion proteins, the zinc finger domain can be N-terminal to the cleavage half-domain, or the cleavage half-domain can be N-terminal to the zinc finger domain. The availability of fusion endonucleases having these different polarities allows targeting (and thereby binding) of zinc finger endonucleases either to opposite strands of the DNA target or to the same strand of the DNA target, thereby increasing the number of possible sequences which can be targeted and cleaved by the fusion proteins.

Claims

1. A method for cleaving cellular chromatin in a region of interest, the method comprising:

(a) selecting the region of interest;

(b) engineering a first zinc finger binding domain to bind to a first nucleotide sequence in the region of interest;

(c) providing a second zinc finger binding domain which binds to a second nucleotide sequence in the region of interest, wherein the second sequence is located between 2 and 50 nucleotides from the first sequence;

(d) expressing a first fusion protein in the cell, the first fusion protein comprising the first zinc finger binding domain and a first cleavage half-domain; and

(e) expressing a second fusion protein in the cell, the second fusion protein comprising the second zinc finger binding domain and a second cleavage half domain;

wherein

(i) the first fusion protein binds to the first nucleotide sequence,

(ii) the second fusion protein binds to the second nucleotide sequence,

(iii) said binding of the first and second fusion proteins positions the cleavage half-domains such that the cellular chromatin is cleaved in the region of interest, and

(iv) in at least one of the first or second fusion proteins, the cleavage half-domain is closer to the N-terminus and the zinc finger binding domain is closer to the C-terminus.

2. The method of claim 1, wherein cleavage occurs between the first and second nucleotide sequences.

3. The method of claim 1 wherein the second zinc finger binding domain is engineered to bind to the second nucleotide sequence.

4. The method of claim 1 wherein the first and second cleavage half-domains are from the same endonuclease.

5. The method of claim 4 wherein the endonuclease is a Type IIS restriction endonuclease.

6. The method of claim 5 wherein the Type IIS restriction endonuclease is FokI.

7. The method of claim 1 wherein the cellular chromatin is in a chromosome.

8. The method of claim 1, wherein the first cleavage half domain is from a Type IIS restriction endonuclease.

9. The method of claim 1, wherein the second cleavage half domain is from a Type IIS restriction endonuclease.

10. The method of claim 1, wherein the first and second nucleotide sequences are on opposite strands of DNA.

11. The method of claim 10, wherein, in the first and second fusion proteins, the cleavage half-domains are closer to the N-termini and the zinc finger binding domains are closer to the C-termini.

12. The method of claim 1, wherein the first and second nucleotide sequences are on the same strand of DNA.

13. The method of claim 12 wherein, in the first fusion protein, the cleavage half-domain is closer to the N-terminus and the zinc finger binding domain is closer to the C-terminus and, in the second fusion protein, the zinc finger binding domain is closer to the N-terminus and the cleavage half-domain is closer to the C-terminus.

14. The method of claim 12 wherein, in the first fusion protein, the zinc finger binding domain is closer to the N-terminus and the cleavage half-domain is closer to the C-terminus and, in the second fusion protein, the cleavage half-domain is closer to the N-terminus and the zinc finger binding domain is closer to the C-terminus.