Patent ID: 8715992

Claim:
A method of preparing at least one I-CreI meganuclease variant having a modified cleavage specificity, said method comprising: (a) replacing amino acids Q44, R68 and/or R70, in reference with SEQ ID NO: 70, with an amino acid selected from the group consisting of A, D, E, G, H, K, N, P, Q, R, S, T and Y to obtain one or more I-CreI meganuclease variants; and (b) selecting said one or more I-CreI meganuclease variants obtained in (a) having at least one of the following R 3 triplet cleaving profile in reference to positions −5 to −3 in a double-strand DNA target, said positions −5 to −3 corresponding to R 3 of the following formula I: (SEQ ID NO: 92) 5′-R 1 CAAAR 2 R 3 R 4 R′ 4 R′ 3 R′ 2 TTTGR′ 1 -3′, wherein: R 1 is absent or present; and when present represents a nucleic acid fragment comprising 1 to 9 nucleotides corresponding either to a random nucleic acid sequence or to a fragment of a I-CreI meganuclease homing site situated from position −20 to −12 (from 5′ to 3′), R 1 corresponding at least to position −12 of said homing site, R 2 represents the nucleic acid doublet ac or ct and corresponds to positions −7 to −6 of said homing site, R 3 represents a nucleic acid triplet corresponding to said positions −5 to −3, selected among g, t, c and a, except the following triplets: gtc, gcc, gtg, gtt and gct, R 4 represents the nucleic acid doublet gt or tc and corresponds to positions −2 to −1 of said homing site, R′ 1 is absent or present; and when present represents a nucleic acid fragment comprising 1 to 9 nucleotides corresponding either to a random nucleic acid sequence or to a fragment of a I-CreI meganuclease homing site situated from position +12 to +20 (from 5′ to 3′), R′ 1 corresponding at least to position +12 of said homing site, R′ 2 represents the nucleic acid doublet ag or gt, and corresponds to positions +6 to +7 of said homing site, R′ 3 represents a nucleic acid triplet corresponding to said positions +3 to +5, selected among g, t, c, and a; R′ 3 being different from gac, ggc, cac, aac, and agc, when R 3 and R′ 3 are non-palindromic, and R′ 4 represents the nucleic acid doublet ga or ac and corresponds to positions +1 to +2 of said homing site.