Patent ID: 8163514

Claim:
A method of cleaving a target recognition site in double-stranded DNA in a non-human cell or an isolated human cell, wherein said target recognition site has a sequence that differs by at least one nucleotide modification from the wild-type I-CreI meganuclease recognition sequence of SEQ ID NO: 4: Position (SEQ ID NO: 4) -9-8-7-6-5-4-3-2-1 5′-C A A A C T G T C G T G A G A C A G T T T G-3′ wherein said target recognition site has a sequence that comprises a substitution at a position corresponding to position −8 of the I-CreI recognition site of SEQ ID NO: 4, wherein the nucleotide corresponding to position −8 of the I-CreI meganuclease recognition sequence of SEQ ID NO: 4 has been altered to a C, the method comprising: (a) introducing into a cell (i) a recombinant meganuclease, or (ii) a nucleic acid that encodes a recombinant meganuclease and causes expression of the recombinant meganuclease in said cell, wherein the recombinant meganuclease comprises a polypeptide having at least 85% sequence identity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO: 1, and which differs from residues 2-153 of the I-CreI meganuclease of SEQ ID NO: 1 by a specificity-altering amino acid substitution corresponding to a substitution in SEQ ID NO: 1 of S32R; and (b) contacting the recombinant meganuclease with the double-stranded DNA, whereby the meganuclease cleaves the target recognition site.