Patent ID: 7914796
Filing Date: 2011-03-29
Classification: C12N

Abstract:
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: 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.