Engineered cleavage half-domains
First Claim
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1. An in vitro 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) in the engineered FokI cleavage half-domain of the first fusion protein the glutamine (Q) residue at position 486 of a wild-type FokI cleavage half-domain is replaced with a glutamic acid (E) residue Q486E) or;
(b) in the engineered FokI cleavage half-domain of the second fusion protein the glutamic acid (E) residue at position 490 of the wild-type FokI cleavage half-domain is replaced with a lysine (K) residue and the isoleucine (I) residue at position 538 of the wild-type FokI cleavage half-domain is replaced with a lysine (K) residue (E490K;
I538K);
(c) in the engineered FokI cleavage half-domain of the third fusion protein the arginine (R) residue at position 487 of the wild-type FokI cleavage half-domain is replaced with an aspartic acid (D) residue (R487D);
(c) in the engineered FokI cleavage half-domain of the fourth fusion protein the aspartic acid (D) residue at position 483 of the wild-type FokI cleavage half-domain is replaced with an arginine (R) residue (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.
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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 these polynucleotides and/or fusion proteins. Also described are methods of using these polypeptides and polynucleotides, for example for targeted cleavage of a genomic sequence.
154 Citations
19 Claims
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1. An in vitro method for cleaving DNA in a cell, the method comprising:
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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) in the engineered FokI cleavage half-domain of the first fusion protein the glutamine (Q) residue at position 486 of a wild-type FokI cleavage half-domain is replaced with a glutamic acid (E) residue Q486E) or; (b) in the engineered FokI cleavage half-domain of the second fusion protein the glutamic acid (E) residue at position 490 of the wild-type FokI cleavage half-domain is replaced with a lysine (K) residue and the isoleucine (I) residue at position 538 of the wild-type FokI cleavage half-domain is replaced with a lysine (K) residue (E490K;
I538K);(c) in the engineered FokI cleavage half-domain of the third fusion protein the arginine (R) residue at position 487 of the wild-type FokI cleavage half-domain is replaced with an aspartic acid (D) residue (R487D); (c) in the engineered FokI cleavage half-domain of the fourth fusion protein the aspartic acid (D) residue at position 483 of the wild-type FokI cleavage half-domain is replaced with an arginine (R) residue (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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification