# US 8,771,986 B2 · Engineered cleavage half-domains

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- Number: US 8,771,986 B2
- Document ID: US8771986B2
- Status: Granted
- Kind: B2
- Application number: 13/239,207
- Filed: 2011-09-21
- Published: 2012-02-16
- Granted: 2014-07-08
- Earliest claimed: 2004-08-06
- Assignees: Sangamo BioSciences, Inc.
- Applicants: Jeffrey C. Miller
- Inventors: Jeffrey C. Miller
- Web page: https://patentgrep.com/patent/US8771986B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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

### Claim 1

A polynucleotide encoding a polypeptide comprising an engineered FokI cleavage half-domain, wherein
(i) the engineered cleavage half-domain comprises a substitution mutation in one or more wild-type amino acid residue 483, 486, 487, 490, 499, 538 or combinations thereof, wherein the amino acid residues are numbered relative to full length wild-type FokI, wherein residues 384 to 579 of wild-type Fold is shown in SEQ ID NO:1;
(ii) the engineered cleavage half-domain fatins an obligate heterodimer with a wild-type cleavage half-domain or a second engineered cleavage half-domain; and
(iii) if the engineered cleavage half-domain comprises a mutation at amino acid residue 490, it further comprises at least one additional amino acid mutation.

### Claim 2 (depends on 1)

The polynucleotide of claim 1, wherein the amino acid residues at positions 490 and 538 are mutated.

### Claim 3 (depends on 2)

The polynucleotide of the claim 2, wherein the engineered cleavage half-domain comprises the polypeptide designated E490K:I538K.

### Claim 4 (depends on 1)

The polynucleotide of claim 1, wherein the wild-type amino acid at position 486 is mutated.

### Claim 5 (depends on 4)

The polynucleotide of claim 4, wherein the engineered cleavage half-domain comprises the polypeptide designated Q486E.

### Claim 6 (depends on 1)

The polynucleotide of claim 1, wherein the wild-type amino acid residue at position 499 is mutated.

### Claim 7 (depends on 6)

The polynucleotide of claim 6, wherein the engineered cleavage half-domain comprises the polypeptide designated I499L.

### Claim 8 (depends on 7)

The polynucleotide of claim 7, wherein the engineered cleavage half-domain comprises the polypeptide designated Q486E:I499L.

### Claim 9 (depends on 1)

The polynucleotide of claim 1 further comprising a sequence encoding a zinc finger protein DNA-binding domain

### Claim 10 (depends on 1)

An isolated cell comprising the polynucleotide of claim 1.

### Claim 11 (depends on 9)

A method for cleaving genomic cellular chromatin in a region of interest, the method comprising:
(a) selecting a first nucleotide sequence in the region if interest;
(b) engineering a first zinc finger binding domain to bind to the first sequence;
(c) expressing a polynucleotide according to claim 9 in a cell, the polynucleotide encoding a first fusion protein comprising a sequence encoding an engineered zinc finger binding domain and the engineered cleavage half-domain;
(d) expressing a second fusion protein in the cell, the second fusion protein comprising a second zinc finger binding domain and a second cleavage half-domain;
wherein the first fusion protein binds to the first nucleotide sequence, the second fusion protein binds to a second nucleotide sequence located between 2 and 50 nucleotides from the first nucleotide sequence, the first and second engineered cleavage domains form a heterodimer that cleaves the cellular chromatin in the region of interest.

### Claim 12 (depends on 11)

The method of claim 11, wherein the second cleavage half-domain is an engineered cleavage half-domain

### Claim 13 (depends on 11)

The method of claim 11, wherein cleavage occurs between the first and second nucleotide sequences.

### Claim 14 (depends on 11)

The method of claim 11, wherein the second zinc finger binding domain is engineered to bind to the second nucleotide sequence.

### Claim 15 (depends on 11)

The method of claim 11, wherein the cellular chromatin is in a chromosome.

### Claim 16 (depends on 11)

The method of claim 11, further comprising contacting the cell with a polynucleotide comprising a third nucleotide sequence, wherein the third nucleotide sequence is homologous but non-identical with the first nucleotide sequence; wherein cleavage of the cellular chromatin in the region of interest facilitates homologous recombination between the first nucleotide sequence and the third nucleotide sequence, resulting in alteration of the first nucleotide sequence.

### Claim 17 (depends on 16)

The method of claim 16, wherein the third nucleotide sequence contains sequences not present in the region of interest that are flanked by sequences homologous to the region of interest.

### Claim 18 (depends on 11)

The method of claim 11, wherein the cell is arrested in the G2 phase of the cell cycle.