Patent ID: 8129134
Filing Date: 2012-03-06
Classification: A61K,A61P,C12N

Abstract:
1. 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: wherein said target recognition site has a sequence that comprises a substitution at a position corresponding to position −6 of the I-CreI recognition site of SEQ ID NO: 4, 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 at least a first specificity-altering amino acid modification at a position corresponding to a position of SEQ ID NO: 1 selected from the group consisting of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 66, 68, 70, 75, 77, 79 and 139; and wherein: (i) if the nucleotide corresponding to position −6 of the I-CreI meganuclease recognition sequence of SEQ ID NO: 4 has been altered to a T, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO: 1 selected from the group consisting of S40C, S40I, S40V, S79A, S79C, S79I, S79V, and K28H; (ii) if the nucleotide corresponding to position −6 of the I-CreI meganuclease recognition sequence has been altered to a C, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO: 1 of K28S; (iii) if the nucleotide corresponding to position −6 of the I-CreI meganuclease recognition sequence of SEQ ID NO: 4 has been altered to a G, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO: 1 selected from the group consisting of S40R and K28S; and (b) contacting the recombinant meganuclease with the double-stranded DNA, whereby the meganuclease cleaves the target recognition site.