I-crei meganuclease variants with modified specificity, method of preparation and uses thereof

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 I-CreI pdb accession code 1g9y, 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 R3 triplet cleaving profile in reference to positions −5 to −3 in a double-strand DNA target, said positions −5 to −3 corresponding to R3 of the following formula I:
• (SEQ ID NO:
• 92)
• 5′-R1CAAAR2R3R4R′4R′3R′2TTTGR′1-3′,
• wherein:
• R1 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′), R1 corresponding at least to position −12 of said homing site,
• R2 represents the nucleic acid doublet ac or ct and corresponds to positions −7 to −6 of said homing site,
• R3 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,
• R4 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 R3 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.