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

Method of preparing a variant of I-CreI meganuclease having specificity modified cleavage, said method comprising: wherein: ES 2347684 T3 (a) replacing Q44, R68 and / or R70 amino acids, referring to the code pdb access lg9y I-CreI, with an amino acid selected from the group consisting of A, D, e, G, H, K, N, P, Q, R, S, T and Y; (B) selecting the variants of I-CreI meganuclease obtained in step (a) having at least one of the following triplet cleaving profile in reference to R3 positions -5 to -3 in a double-stranded DNA target, corresponding said positions -5 to -3 to R3 of the following formula I: 5'- R1CAAAR2R3R4R'4R'3R'2TTTGR'1, 3 ', R1 is absent or present; and when present represents a nucleic acid fragment comprising nucleotides 1 to 9 corresponding to a random nucleic acid sequence or fragment site directed movement of I-CreI meganuclease situated from position -20 to -12 (from 5 'to 3'), at least R1 corresponding to the -12 position of said site-directed movement, R2 represents doublet ac or ct nucleic acid 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, cya, 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 site-directed movement, R.sub.1 is absent or present; and when present represents a nucleic acid fragment comprising nucleotides 1 to 9 corresponding to a random nucleic acid sequence or a fragment of a site directed movement of I-CreI meganuclease located from +12 to +20 position ( 5 'to 3'), R '1 corresponding at least to the +12 position of said site-directed movement, R'2 represents the nucleic acid doublet ag or gt, and corresponds to positions +6 to +7 of the site directed motion, R'3 represents a nucleic acid triplet corresponding to said positions +3 to +5, selected among g, t, cya; R'3 being different from gac, GGC, cac, aac, and agc, when R3 and R'3 are not palindromic, R '4 represents the nucleic acid doublet ga or ac and corresponds to positions +1 to +2 of said site directed motion.