Patent ID: 8377674

Claim:
A method for producing a genetically-modified eukaryotic non-human cell or an isolated human cell by modifying a target nucleic acid in a chromosome of the cell, the method comprising: introducing into the cell: (i) a recombinant meganuclease, or (ii) a first nucleic acid that encodes a recombinant meganuclease and causes expression of the recombinant meganuclease in the 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 one 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 the at least one specificity-altering amino acid modification is 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, Q381, 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, R70S, D75C, D75E, D75H, D75L, D75Q, D75R, D75Y, I77E, I77Q, I77R, I77S, S79A, S79C, S79I, S79V, K139H and K139Y; wherein the DNA-binding affinity of the recombinant meganuclease has been altered compared to the DNA-binding affinity of the wild-type I-CreI meganuclease of SEQ ID NO: 1, and wherein the polypeptide further comprises at least one modification affecting DNA-binding affinity corresponding to a substitution in the polypeptide of SEQ ID NO: 1 selected from the group consisting of: P29D, P29E, P29H, P29K, P29N, P29Q, P29R, P29S, P29T, K34D, K34E, K34H, K34N, K34Q, K34S, K34T, K48D, K48E, K48H, K48N, K48Q, K48S, K48T, R51D, R51E, R51H, R51N, R51Q, R51S, R51T, V64D, V64E, V64H, V64K, V64N, V64Q, V64R, V64S, V64T, E80H, E80K, E80N, E80Q, E80R, E80S, E80T, I81D, I81E, I81H, I81K, I81N, I81Q, I81R, I81S, I81T, K82D, K82E, K82H, K82N, K82Q, K82S, K82T, L112D, L112E, L112H, L112K, L112N, L112Q, L112R, L112S, L112T, K116D, K116E, K116H, K116N, K116Q, K116S, K116T, D137H, D137K, D137N, D137Q, D137R, D137S, D137T, K139D, K139E, K139H, K139N, K139Q, K139S, K139T, T143D, T143E, T143K and T143R; and wherein the recombinant meganuclease cleaves a site in the target nucleic acid in the chromosome and the target nucleic acid is modified by at least one base insertion, at least one base deletion, or at least one frameshift mutation in the chromosome.