Methods of cleaving DNA with rationally-designed meganucleases

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 in the wild-type I-CreI meganuclease recognition sequence of SEQ ID NO:

• 4:
• Position −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′ (SEQ ID NO:
• 4)
• wherein said target recognition site has a sequence that comprises a substitution at a position corresponding to position −5 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;
• wherein:
• (i) if the nucleotide corresponding to position −5 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 A42Q, A42R, K28C, Y66M and Y66K;
• (ii) if the nucleotide corresponding to position −5 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 K28C, A42Q, Y66M, and Y66K;
• (iii) if the nucleotide corresponding to position −5 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 Y66M, and Y66K;
• and
• wherein:
• the recombinant meganuclease comprises at least a second specificity-altering amino acid modification corresponding to a substitution in SEQ ID NO:
• 1 selected from the group consisting of:
• I24C, I24K, I24R, Q26A, Q26E, Q26K, Q26S, K28A, K28C, K28H, K28Q, K28R, K28S, N30E, N30K, N30Q, N30R, S32A, S32C, S32D, S32E, S32H, S32I, S32K, S32L, S32N, S32Q, S32R, S32T, S32V, Y33C, Y33D, Y33E, Y33F, Y33H, Y33I, Y33L, Y33R, Y33V, Q38C, Q38E, Q38H, Q38I, Q38K, Q38L, Q38N, Q38R, S40A, S40C, S40E, S40I, S40Q, S40R, S40V, A42E, A42Q, A42R, Q44A, Q44C, Q44D, Q44E, Q44I, Q44K, Q44L, Q44N, Q44R, Q44T, Q44V, T46A, T46C, T46D, T46E, T46G, T46H, T46K, T46Q, T46R, Y66K, Y66M, R68C, R68E, R68F, R68H, R68K, R68L, R68M, R68Q, R68Y, R70A, R70C, R70D, R70E, R70G, R70H, R70K, R70L, R70Q, R705, D75C, D75E, D75H, D75L, D75Q, D75R, D75Y, I77E, I77Q, I77R, I77S, S79A, S79C, S791, S79V, K139H and K139Y;
• and
• (b) contacting the recombinant meganuclease with the double-stranded DNA, whereby the meganuclease cleaves the target recognition site.