Patent ID: 8124369
Filing Date: 2012-02-28
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 −9 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; wherein: (ii) if the nucleotide corresponding to position −9 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 S32L, S32V, S32C, S32D, S32I, S32N, S32H, S32Q and S32T; (iii) if the nucleotide corresponding to position −9 of the I-CreI meganuclease recognition sequence of SEQ ID NO: 4 has been altered to an A, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO: 1 selected from the group consisting of S32N, S32H, S32Q and S32T; and (b) contacting the recombinant meganuclease with the double-stranded DNA, whereby the meganuclease cleaves the target recognition sequence.