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

Abstract

Method of preparing I-CreI meganuclease variants having a modified cleavage specificity, variants obtainable by said method and their applications either for cleaving new DNA target or for genetic engineering and genome engineering for non-therapeutic purposes. Nucleic acids encoding said variants, expression cassettes comprising said nucleic acids, vectors comprising said expression cassettes, cells or organisms, plants or animals except humans, transformed by said vectors.

1 . 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 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:   
       
         
           
                 
                 
               
                   5′-R 1 CAAAR 2 R 3 R 4 R′ 4 R′ 3 R′ 2 TTTGR′ 1 -3′, 
                   (SEQ ID NO: 92) 
                 
             
                
               
            
           
         
         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 get, 
         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 age, 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. 
       
     
     
         2 . The method according to  claim 1 , wherein said nucleic acid triplet R 3  is selected among the following triplets: ggg, gga, ggt, ggc, gag, gaa, gat, gac, gta, gcg, gca, tgg, tga, tgt, tgc, tag, taa, tat, tac, ttg, tta, ttt, ttc, tcg, tca, tct, tcc, agg, aga, agt, agc, aag, aaa, aat, aac, atg, ata, att, atc, acg, aca, act, acc, cgg, cga, cgt, cgc, cag, caa, cat, cac, ctg, eta, ctt, ctc, ccg, cca, cct and ccc. 
     
     
         3 . The method according to  claim 2 , wherein said nucleic acid triplet R 3  is selected among the following triplets: ggg, ggt, ggc, gag, gat, gac, gta, gcg, gca, tag, taa, tat, tac, ttg, ttt, ttc, tcg, tct, tee, agg, aag, aat, aac, att, atc, act, acc, cag, cat, cac, ctt, ctc, ccg, cct and ccc. 
     
     
         4 . The method according to  claim 1 , wherein the at least one I-CreI meganuclease variant selected in (b) is selected from the group consisting of: A44/A68/A70, A44/A68/G70, A44/A68/H70, A44/A68/K70, A44/A68/N70, A44/A68/Q70, A44/A68/R70, A44/A68/S70, A44/A68/T70, A44/D68/H70, A44/D68/K70, A44/D68/R70, A44/G68/H70, A44/G68/K70, A44/G68/N70, A44/G68/P70, A44/G68/R70, A44/H68/A70, A44/H68/G70, A44/H68/H70, A44/H68/K70, A44/H68/N70, A44/H68/Q70, A44/H68/R70, A44/H68/S70, A44/H68/T70, A44/K68/A70, A44/K68/G70, A44/K68/H70, A44/K68/K70, A44/K68/N70, A44/K68/Q70, A44/K68/R70, A44/K68/S70, A44/K68/T70, A44/N68/A70, A44/N68/E70, A44/N68/G70, A44/N68/H70, A44/N68/K70, A44/N68/N70, A44/N68/Q70, A44/N68/R70, A44/N68/S70, A44/N68/T70, A44/Q68/A70, A44/Q68/D70, A44/Q68/G70, A44/Q68/H70, A44/Q68/N70, A44/Q68/R70, A44/Q68/S70, A44/R68/A70, A44/R68/D70, A44/R68/E70, A44/R68/G70, A44/R68/H70, A44/R68/K70, A44/R68/L70, A44/R68/N70, A44/R68/R70, A44/R68/S70, A44/R68/T70, A44/S68/A70, A44/S68/G70, A44/S68/K70, A44/S68/N70, A44/S68/Q70, A44/S68/R70, A44/S68/S70, A44/S68/T70, A44/T68/A70, A44/T68/G70, A44/T68/H70, A44/T68/K70, A44/T68/N70, A44/T68/Q70, A44/T68/R70, A44/T68/S70, A44/T68/T70, D44/D68/H70, D44/N68/S70, D44/R68/A70, D44/R68/K70, D44/R68/N70, D44/R68/Q70, D44/R68/R70, D44/R68/S70, D44/R68/T70, E44/H68/H70, E44/R68/A70, E44/R68/H70, E44/R68/N70, E44/R68/S70, E44/R68/T70, E44/S68/T70, G44/H68/K70, G44/Q68/H70, G44/R68/Q70, G44/R68/R70, G44/T68/D70, G44/T68/P70, G44/T68/R70, H44/A68/S70, H44/A68/T70, H44/R68/A70, H44/R68/D70, H44/R68/E70, H44/R68/G70, H44/R68/N70, H44/R68/R70, H44/R68/S70, H44/R68/T70, H44/S68/G70, H44/S68/S70, H44/S68/T70, H44/T68/S70, H44/T68/T70, K44/A68/A70, K44/A68/D70, K44/A68/E70, K44/A68/G70, K44/A68/H70, K44/A68/N70, K44/A68/Q70, K44/A68/S70, K44/A68/T70, K44/D68/A70, K44/D68/T70, K44/E68/G70, K44/E68/N70, K44/E68/S70, K44/G68/A70, K44/G68/G70, K44/G68/N70, K44/G68/S70, K44/G68/T70, K44/H68/D70, K44/H68/E70, K44/H68/G70, K44/H68/N70, K44/H68/S70, K44/H68/T70, K44/K68/A70, K44/K68/D70, K44/K68/H70, K44/K68/T70, K44/N68/A70, K44/N68/D70, K44/N68/E70, K44/N68/G70, K441N68/H70, K44/N68/N70, K44/N68/Q70, K44/N68/S70, K44/N68/T70, K44/P68/H70, K44/Q68/A70, K44/Q68/D70, K44/Q68/E70, K44/Q68/S70, K44/Q68/T70, K44/R68/A70, K44/R68/D70, K44/R68/E70, K44/R68/G70, K44/R68/H70, K44/R68/N70, K44/R68/Q70, K44/R68/S70, K44/R68/T70, K44/S68/A70, K44/S68/D70, K44/S68/H70, K44/S68/N70, K44/S68/S70, K44/S68/T70, K44/T68/A70, K44/T68/D70, K44/T68/E70, K44/T68/G70, K44/T68/H70, K44/T68/N70, K44/T68/Q70, K44/T68/S70, K44/T68/T70, N44/A68/H70, N44/A68/R70, N44/H68/N70, N44/H68/R70, N44/K68/G70, N44/K68/H70, N44/K68/R70, N44/K68/S70, N44/N68/R70, N44/P68/D70, N44/Q68/H70, N44/Q68/R70, N44/R68/A70, N44/R68/D70, N44/R68/E70, N44/R68/G70, N44/R68/H70, N44/R68/K70, N44/R68/N70, N44/R68/R70, N44/R68/S70, N44/R68/T70, N44/S68/G70, N44/S68/H70, N44/S68/K70, N44/S68/R70, N44/T68/H70, N44/T68/K70, N44/T68/Q70, N44/T68/R70, N44/T68/S70, P44/N68/D70, P44/T68/T70, Q44/A68/A70, Q44/A68/H70, Q44/A68/R70, Q44/G68/K70, Q44/G68/R70, Q44/K68/G70, Q44/N68/A70, Q44/N68/H70, Q44/N68/S70, Q44/P68/P70, Q44/Q68/G70, Q44/R68/A70, Q44/R68/D70, Q44/R68/E70, Q44/R68/G70, Q44/R68/H70, Q44/R68/N70, Q44/R68/Q70, Q44/R68/S70, Q44/S68/H70, Q44/S68/R70, Q44/S68/S70, Q44/T68/A70, Q44/T68/G70, Q44/T68/H70, Q44/T68/R70, R44/A68/G70, R44/A68/T70, R44/G68/T70, R44/H68/D70, R44/H68/T70, R44/N68/T70, R44/R68/A70, R44/R68/D70, R44/R68/E70, R44/R68/G70, R44/R68/N70, R44/R68/Q70, R44/R68/S70, R44/R68/T70, R44/S68/G70, R44/S68/N70, R44/S68/S70, R44/S68/T70, S44/D68/K70, S44/H68/R70, S44/R68/G70, S44/R68/N70, S44/R68/R70, S44/R68/S70, T44/A68/K70, T44/A68/R70, T44/H68/R70, T44/K68/R70, T44/N68/P70, T44/N68/R70, T44/Q68/K70, T44/Q68/R70, T44/R68/A70, T44/R68/D70, T44/R68/E70, T44/R68/G70, T44/R68/H70, T44/R68/K70, T44/R68/N70, T44/R68/Q70, T44/R68/R70, T44/R68/S70, T44/R68/T70, T44/S68/K70, T44/S68/R70, T44/T68/K70, and T44/T68/R70. 
     
     
         5 . The method according to  claim 1 , wherein said selecting (b) of said at least one I-CreI meganuclease variant is performed in vivo in yeast cells. 
     
     
         6 . At least one I-CreI meganuclease variant prepared by the method according to  claim 1 , wherein said at least one I-CreI meganuclease variant is selected from the group consisting of: A44/A68/A70, A44/A68/G70, A44/A68/H70, A44/A68/K70, A44/A68/N70, A44/A68/Q70, A44/A68/S70, A44/A68/T70, A44/D68/H70, A44/D68/K70, A44/D68/R70, A44/G68/H70, A44/G68/K70, A44/G68/N70, A44/G68/P70, A44/H68/A70, A44/H68/G70, A44/H68/H70, A44/H68/K70, A44/H68/N70, A44/H68/Q70, A44/H68/S70, A44/H68/T70, A44/K68/A70, A44/K68/G70, A44/K68/H70, A44/K68/N70, A44/K68/Q70, A44/K68/R70, A44/K68/S70, A44/K68/T70, A44/N68/A70, A44/N68/E70, A44/N68/G70, A44/N68/H70, A44/N68/K70, A44/N68/N70, A44/N68/Q70, A44/N68/R70, A44/N68/S70, A44/N68/T70, A44/Q68/A70, A44/Q68/D70, A44/Q68/G70, A44/Q68/H70, A44/Q68/N70, A44/Q68/S70, A44/R68/E70, A44/R68/K70, A44/R68/L70, A44/S68/A70, A44/S68/G70, A44/S68/N70, A44/S68/Q70, A44/S68/R70, A44/S68/S70, A44/S68/T70, A44/T68/A70, A44/T68/G70, A44/T68/H70, A44/T68/N70, A44/T68/Q70, A44/T68/S70, A44/T68/T70, D44/D68/H70, D44/N68/S70, D44/R68/A70, D44/R68/N70, D44/R68/Q70, D44/R68/R70, D44/R68/S70, D44/R68/T70, E44/H68/H70, E44/R68/A70, E44/R68/H70, E44/R68/N70, E44/R68/S70, E44/R68/T70, E44/S68/T70, G44/H68/K70, G44/Q68/H70, G44/R68/Q70, G44/T68/D70, G44/T68/P70, G44/T68/R70, H44/A68/S70, H44/A68/T70, H44/R68/D70, H44/R68/E70, H44/R68/G70, H44/R68/N70, H44/R68/R70, H44/R68/S70, H44/S68/G70, H44/S68/S70, H44/S68/T70, H44/T68/S70, H44/T68/T70, K44/A68/A70, K44/A68/D70, K44/A68/E70, K44/A68/G70, K44/A68/H70, K44/A68/N70, K44/A68/Q70, K44/D68/A70, K44/D68/T70, K44/E68/G70, K44/E68/S70, K44/G68/A70, K44/G68/G70, K44/G68/N70, K44/G68/S70, K44/G68/T70, K44/H68/D70, K44/H68/E70, K44/H68/G70, K44/H68/N70, K44/H68/S70, K44/H68/T70, K44/K68/A70, K44/K68/D70, K44/K68/H70, K44/K68/T70, K44/N68/A70, K44/N68/D70, K44/N68/E70, K44/N68/G70, K44/N68/H70, K44/N68/N70, K44/N68/Q70, K44/N68/S70, K44/N68/T70, K44/P68/H70, K44/Q68/A70, K44/Q68/D70, K44/Q68/E70, K44/Q68/S70, K44/Q68/T70, K44/R68/A70, K44/R68/D70, K44/R68/E70, K44/R68/G70, K44/R68/H70, K44/R68/N70, K44/R68/S70, K44/S68/A70, K44/S68/D70, K44/S68/H70, K44/S68/N70, K44/S68/S70, K44/S68/T70, K44/T68/A70, K44/T68/D70, K44/T68/E70, K44/T68/G70, K44/T68/H70, K44/T68/N70, K44/T68/Q70, K44/T68/S70, K44/T68/T70, N44/A68/H70, N44/H68/N70, N44/H68/R70, N44/K68/G70, N44/K68/H70, N44/K68/R70, N44/K68/S70, N44/P68/D70, N44/Q68/H70, N44/R68/A70, N44/R68/D70, N44/R68/E70, N44/R68/K70, N44/S68/G70, N44/S68/H70, N44/S68/K70, N44/S68/R70, N44/T68/H70, N44/T68/K70, N44/T68/Q70, N44/T68/S70, P44/N68/D70, P44/T68/T70, Q44/G68/K70, Q44/G68/R70, Q44/K68/G70, Q44/N68/A70, Q44/N68/H70, Q44/N68/S70, Q44/P68/P70, Q44/Q68/G70, Q44/R68/D70, Q44/R68/E70, Q44/R68/G70, Q44/R68/Q70, Q44/S68/S70, Q44/T68/A70, Q44/T68/G70, Q44/T68/H70, R44/A68/G70, R44/A68/T70, R44/G68/T70, R44/H68/D70, R44/H68/T70, R44/N68/T70, R44/R68/A70, R44/R68/D70, R44/R68/E70, R44/R68/G70, R44/R68/Q70, R44/R68/S70, R44/R68/T70, R44/S68/G70, R44/S68/N70, R44/S68/S70, R44/S68/T70, S44/D68/K70, S44/R68/R70, S44/R68/S70, T44/A68/K70, T44/N68/P70, T44/N68/R70, T44/R68/E70, T44/R68/Q70, and T44/S68/K70. 
     
     
         7 . The at least one I-CreI meganuclease variant according to  claim 6 , wherein said at least one I-CreI meganuclease variant comprises an alanine (A) or an asparagine (N) in position 44 and cleaves a double-strand nucleic acid target comprising nucleotide a in position −4, and/or nucleotide t in position +4. 
     
     
         8 . The at least one I-CreI meganuclease variant according to  claim 6 , wherein said at least one I-CreI meganuclease variant comprises a lysine (K) in position 44 and cleaves a double-strand nucleic acid target comprising nucleotide c in position −4, and/or nucleotide g in position +4. 
     
     
         9 . The at least one I-CreI meganuclease variant according to  claim 6 , wherein said at least one I-CreI meganuclease variant comprises a glutamine (Q) in position 44 and cleaves a double-strand nucleic acid target comprising nucleotide t in position −4, and/or nucleotide a in position +4. 
     
     
         10 . The at least one I-CreI meganuclease variant according to  claim 6 , wherein said at least one I-CreI meganuclease variant is a homodimer. 
     
     
         11 . The at least one I-CreI meganuclease variant according to  claim 6 , wherein said at least one I-CreI meganuclease variant is a heterodimer consisting of two monomers, each of said monomers being selected from a different I-CreI meganuclease variant as defined in  claim 6 . 
     
     
         12 . A polynucleotide encoding the at least one I-CreI meganuclease variant according to  claim 6 . 
     
     
         13 . An expression cassette s  comprising the polynucleotide according to  claim 12  and regulation sequences. 
     
     
         14 . An expression vector comprising the expression cassette according to  claim 13 . 
     
     
         15 . The expression vector according to  claim 14 , wherein said expression vector further comprises a targeting DNA construct. 
     
     
         16 . The expression vector according to  claim 15 , wherein said targeting DNA construct comprises a sequence sharing homologies with a region surrounding a cleavage site of the at least one I-CreI meganuclease variant. 
     
     
         17 . The expression vector according to  claim 16 , wherein said targeting DNA construct comprises:
 a) sequences sharing homologies with the region surrounding the cleavage site of the at least one I-CreI meganuclease variant, and   b) sequences to be introduced flanked by sequence as in a).   
     
     
         18 . A modified cell comprising the polynucleotide according to  claim 12 . 
     
     
         19 . A transgenic plant comprising the polynucleotide according to  claim 12 . 
     
     
         20 . A non-human transgenic mammal comprising the polynucleotide according to  claim 12 . 
     
     
         21 . A method of genetic engineering comprising double-strand nucleic acid breaking in a site of interest located on a vector, comprising a DNA target of at least one I-CreI meganuclease variant according to  claim 6 , by contacting said vector with said at least one I-CreI meganuclease variant, thereby inducing a homologous recombination with another vector presenting homology with a sequence surrounding a cleavage site of said at least one I-CreI meganuclease variant. 
     
     
         22 . A method of genome engineering comprising:
 1) double-strand breaking a genomic locus comprising at least one recognition and cleavage site of at least one I-CreI meganuclease variant according to  claim 6 , by contacting said cleavage site with said at least one I-CreI meganuclease variant; and   2) maintaining said broken genomic locus under conditions appropriate for homologous recombination with a targeting DNA construct comprising a sequence to be introduced in said genomic locus, flanked by sequences sharing homologies with said genomic locus.   
     
     
         23 . A method of genome engineering comprising:
 1) double-strand breaking a genomic locus comprising at least one recognition and cleavage site of at least one I-CreI meganuclease variant according to  claim 6 , by contacting said cleavage site with said at least one I-CreI meganuclease variant; and   2) maintaining said broken genomic locus under conditions appropriate for homologous recombination with a chromosomal DNA sharing homologies to regions surrounding said cleavage site.   
     
     
         24 . A composition comprising said at least one I-CreI meganuclease variant according to  claim 6 . 
     
     
         25 . The composition according to  claim 24 , wherein said composition further comprises a targeting DNA construct comprising a sequence which repairs a site of interest flanked by sequences sharing homologies with a targeted locus. 
     
     
         26 . A modified cell comprising the expression vector according to  claim 14 . 
     
     
         27 . A transgenic plant comprising the expression vector according to  claim 14 . 
     
     
         28 . A non-human transgenic mammal comprising the expression vector according to  claim 14 . 
     
     
         29 . A composition comprising said polynucleotide according to  claim 12 . 
     
     
         30 . The composition according to  claim 29 , wherein said composition further comprises a targeting DNA construct comprising a sequence which repairs a site of interest flanked by sequences sharing homologies with a targeted locus. 
     
     
         31 . A composition comprising said expression vector according to  claim 14 . 
     
     
         32 . The composition according to  claim 31 , wherein said composition further comprises a targeting DNA construct comprising a sequence which repairs a site of interest flanked by sequences sharing homologies with a targeted locus.