Patent ID: 8771986

Claim:
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.