Custom-made meganuclease and use thereof

Abstract

New rare-cutting endonucleases, also called custom-made meganucleases, which recognize and cleave a specific nucleotide sequence, derived polynucleotide sequences, recombinant vector cell, animal, or plant comprising said polynucleotide sequences, process for producing said rare-cutting endonucleases and any use thereof, more particularly, for genetic engineering, antiviral therapy and gene therapy.

1 - 40 . (canceled) 
     
     
         41 . A method for producing LAGLIDADG meganuclease variants derived from an initial LAGLIDADG meganuclease, said meganuclease variants being able to cleave a desired DNA target which is different from the DNA target of the initial LAGLIDADG meganuclease, wherein said method comprises:
 a) generating a combinatorial library of genes coding for LAGLIDADG meganuclease variants by introducing diversity into several codons of a gene encoding said initial LAGLIDADG meganuclease, wherein said codons code for amino acid residues of the four-stranded beta-sheet of one domain or one subunit of said initial LAGLIDADG meganuclease that interact directly or indirectly with different bases of the DNA target of said initial meganuclease;   b) screening meganuclease variants encoded by the genes of the combinatorial library obtained in a) by an in vivo cleavage assay in an isolated yeast cell or an isolated mammalian cell, under conditions where a double-strand break in the DNA target which is generated by said variants leads to the activation of a positive selection marker or a reporter gene, or the inactivation of a negative selection marker or a reporter gene, by recombination-mediated repair of said DNA double-strand break, and   c) isolating the genes encoding the meganuclease variants obtained in b) which are able to cleave said DNA target.   
     
     
         42 . The method according to  claim 41 , wherein said amino acid residues that interact directly or indirectly with the DNA target of the initial meganuclease are selected from the group consisting of: D, E, H, K, N, Q, R, S, T and Y. 
     
     
         43 . The method according to  claim 42 , wherein said amino acid residues are substituted with an amino acid selected from the group consisting of: A, D, E, G, H, K, N, P, Q, S and T. 
     
     
         44 . The method according to  claim 41 , wherein said initial LAGLIDADG meganuclease is a natural or a modified meganuclease. 
     
     
         45 . The method according to  claim 41 , wherein said initial LAGLIDADG meganuclease is selected from the group consisting of: I-Cre I, I-Dmo I, PI-Sce I, and PI-Pfu I. 
     
     
         46 . The method according to  claim 45 , wherein said initial LAGLIDADG meganuclease is I-Cre I. 
     
     
         47 . The method according to claim, 46 wherein I-Cre I variants are prepared by introducing amino acid substitutions in at least three positions of the I-CreI of SEQ ID NO: 10, wherein said positions are selected from the group consisting of Q26, K28, N30, S32, Y33, Q38, Q44, R68 and R70 of SEQ ID NO:10. 
     
     
         48 . The method according to  claim 47 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, N30, Y33, Q38, Q44, R68 and R70. 
     
     
         49 . The method according to  claim 47 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, N30, Y33, Q44, R68 and R70. 
     
     
         50 . The method according to  claim 47 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, Y33, Q38, Q44, R68 and R70. 
     
     
         51 . The method according to  claim 47 , further introducing an amino acid substitution with an uncharged amino acid, at position 75 of the I-Cre I of SEQ ID NO: 10. 
     
     
         52 . The method according to  claim 51 , wherein said uncharged amino acid is an asparagine residue. 
     
     
         53 . The method according to  claim 41 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprise an expression cassette comprising said desired DNA target and the coding region of an active negative selection marker, the coding region of an inactive positive selection marker, or the coding region of an active or inactive reporter gene product. 
     
     
         54 . The method according to  claim 41 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprises an expression cassette comprising (i) said desired DNA target and a polynucleotide comprising two direct repeats of an internal portion of a polynucleotide encoding an inactive positive selection marker, wherein the two repeats are separated by the desired DNA target, or (ii) said desired DNA target and a polynucleotide comprising two direct repeats of an internal portion of an inactive reporter gene, wherein the two repeats are separated by the desired DNA target. 
     
     
         55 . The method according to  claim 53 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprise an expression cassette comprising said desired DNA target and (i) a polynucleotide encoding an inactive positive selection marker due to the presence of a mutation or a deletion, or (ii) an inactive reporter gene due to the presence of a mutation or a deletion, wherein said desired DNA target is inserted in (i) or (ii) at the place of the deletion or in the vicinity of the mutation, and wherein the isolated yeast or mammalian cell further comprises a genetic construct comprising the segment of the polynucleotide encoding the positive selection marker which has been deleted or mutated, or the segment of the reporter gene which has been deleted or mutated, flanked at each side by the fragments of the polynucleotide encoding the positive selection marker or the reporter gene bordering the deletion/insertion. 
     
     
         56 . The method according to  claim 41 , wherein the double-strand break in the desired DNA target is repaired by homologous recombination between two direct repeats. 
     
     
         57 . The method according to  claim 41 , wherein the double-strand break in the desired DNA target is repaired by gene conversion. 
     
     
         58 . The method according to  claim 53 , wherein the expression cassette is inserted in a plasmid which is maintained in an episomal form in said isolated yeast or mammalian cell. 
     
     
         59 . The method according to  claim 53 , wherein the expression cassette is integrated in the chromosome of said isolated yeast or mammalian cell. 
     
     
         60 . The method according to  claim 41 , wherein said positive selection marker is an antibiotic resistance or an auxotrophy marker. 
     
     
         61 . The method according to  claim 53 , wherein said expression cassette comprises a additional selection marker. 
     
     
         62 . A method for producing LAGLIDADG meganuclease variants derived from an initial LAGLIDADG meganuclease, said meganuclease variants being able to cleave a desired DNA target which is different from the DNA target of the initial LAGLIDADG meganuclease, wherein said method comprises:
 a) generating a combinatorial library of genes coding for LAGLIDADG meganuclease variants by introducing diversity into several codons of a gene encoding said initial LAGLIDADG meganuclease, wherein said codons code for amino acid residues of the four-stranded beta-sheet of one domain or one subunit of said initial LAGLIDADG meganuclease that interact directly or indirectly with different bases of the DNA target of said initial meganuclease;   b) selecting meganuclease variants encoded by the genes of the combinatorial library that are able to cleave said desired DNA target, by an in vivo cleavage assay in an isolated yeast cell or an isolated mammalian cell, under conditions where a double-strand break in the desired DNA target which is generated by said variant leads to the activation of a positive selection marker, or the inactivation of a negative selection marker, by recombination-mediated repair of said desired DNA double-strand break, in the isolated yeast cell or the isolated mammalian cell, and recovering a subset of genes encoding the meganuclease variants able to cleave said desired DNA target;   c) screening meganuclease variants encoded by the subset of genes obtained in b) by an in vivo cleavage assay in an isolated yeast cell or an isolated mammalian cell, under conditions where a double-strand break in the desired DNA target which is generated by said variant leads to the activation of a positive selection marker or a reporter gene, or the inactivation of a negative selection marker or a reporter gene, by recombination-mediated repair of said desired DNA double-strand break, and recovering the subset of meganuclease variants able to cleave said desired DNA target;   d) isolating the genes encoding the meganuclease variants obtained in c) and able to cleave said desired DNA target.   
     
     
         63 . The method according to  claim 62 , wherein said amino acid residues that interact directly or indirectly with the DNA target of the initial meganuclease are selected from the group consisting of: D, E, H, K, N, Q, R, S, T and Y. 
     
     
         64 . The method according to  claim 63 , wherein said amino acid residues are substituted with an amino acid selected from the group consisting of: A, D, E, G, H, K, N, P, Q, S and T. 
     
     
         65 . The method according to  claim 62 , wherein said initial LAGLIDADG meganuclease is a natural or a modified meganuclease. 
     
     
         66 . The method according to  claim 62 , wherein said initial LAGLIDADG meganuclease is selected from the group consisting of: I-Cre I, I-Dmo I, PI-Sce I, and PI-Pfu I. 
     
     
         67 . The method according to  claim 66 , wherein said initial LAGLIDADG meganuclease is I-Cre I. 
     
     
         68 . The method according to  claim 67 , wherein I-Cre I variants are prepared by introducing amino acid substitutions in at least three positions of the I-CreI of SEQ ID NO: 10, wherein said positions are selected from the group consisting of Q26, K28, N30, S32, Y33, Q38, Q44, R68 and R70 of SEQ ID NO:10. 
     
     
         69 . The method according to  claim 68 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, N30, Y33, Q40, Q44, R68 and R70. 
     
     
         70 . The method according to  claim 68 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, N30, Y33, Q44, R68 and R70. 
     
     
         71 . The method according to  claim 68 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, Y33, Q38, Q44, R68 and R70. 
     
     
         72 . The method according to  claim 68 , further introducing an amino acid substitution with an uncharged amino acid, at position 75 of the I-Cre I of SEQ ID NO: 10. 
     
     
         73 . The method according to  claim 72 , wherein said uncharged amino acid is an asparagine residue. 
     
     
         74 . The method according to  claim 62 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprise an expression cassette comprising said desired DNA target and the coding region of an active negative selection marker, the coding region of an inactive positive selection marker, or the coding region of an active or inactive reporter gene. 
     
     
         75 . The method according to  claim 62 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprises an expression cassette comprising (i) said desired DNA target and a polynucleotide comprising two direct repeats of an internal portion of a polynucleotide encoding an inactive positive selection marker, wherein the two repeats are separated by the desired DNA target, or (ii) said desired DNA target and a polynucleotide comprising two direct repeats of an internal portion of an inactive reporter gene, wherein the two repeats are separated by the desired DNA target. 
     
     
         76 . The method according to  claim 74 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprise an expression cassette comprising said desired DNA target and (i) a polynucleotide encoding an inactive positive selection marker due to the presence of a mutation or a deletion, or (ii) an inactive reporter gene due to the presence of a mutation or a deletion, wherein said desired DNA target is inserted in (i) or (ii) at the place of the deletion or in the vicinity of the mutation, and wherein the isolated yeast or mammalian cell further comprises a genetic construct comprising the segment of the polynucleotide encoding the positive selection marker which has been deleted or mutated, or the segment of the reporter gene which has been deleted or mutated, flanked at each side by the fragments of the polynucleotide encoding the positive selection marker or the reporter gene bordering the deletion/insertion. 
     
     
         77 . The method according to  claim 62 , wherein the double-strand break in the desired DNA target is repaired by homologous recombination between two direct repeats. 
     
     
         78 . The method according to  claim 62 , wherein the double-strand break in the DNA target is repaired by gene conversion. 
     
     
         79 . The method according to  claim 74 , wherein the expression cassette is inserted in a plasmid which is maintained in an episomal form in said isolated yeast or mammalian cell. 
     
     
         80 . The method according to  claim 74 , wherein the expression cassette is integrated in the chromosome of said isolated yeast or mammalian cell. 
     
     
         81 . The method according to  claim 62 , wherein said positive selection marker is an antibiotic resistance or an auxotrophy marker. 
     
     
         82 . The method according to  claim 74 , wherein said expression cassette comprises an additional selection marker. 
     
     
         83 . A method for producing LAGLIDADG meganuclease variants derived from an initial LAGLIDADG meganuclease, said meganuclease variants being able to cleave a desired DNA target which is different from the DNA target of the initial LAGLIDADG meganuclease, wherein said method comprises:
 a) generating a combinatorial library of genes coding for LAGLIDADG meganuclease variants by introducing diversity into several codons of a gene encoding said initial LAGLIDADG meganuclease, wherein said codons code for amino acid residues of the four-stranded beta-sheet of one domain or one subunit of said initial meganuclease that interact directly or indirectly with different bases of the DNA target of said initial meganuclease;   b) selecting meganuclease variants encoded by the genes of the combinatorial library that are able to bind said desired DNA target in vitro, under conditions excluding the presence of manganese or magnesium by incubating said meganuclease variants with said DNA target immobilized on a support and recovering only the subset of bound meganuclease variants;   c) screening the subset of bound meganuclease variants obtained in b) by a binding assay consisting of incubation of said selected meganuclease variants with said desired DNA target immobilized on a support and recovering a subset of bound meganuclease variants;   d) selecting among the meganuclease variants recovered in step c), the variants able to cleave said desired DNA target, by an in vivo cleavage assay, in an isolated yeast cell or an isolated mammalian cell, under conditions where the double-strand break in the desired DNA target which is generated by said variant leads to the activation of a positive selection marker, or the inactivation of a negative selection marker, by recombination-mediated repair of said DNA double-strand break, and recovering a subset of meganuclease variants able to cleave said desired DNA target;   e) screening said subset of meganuclease variants obtained in d) by an in vivo cleavage assay in an isolated yeast cell or a mammalian cell, under conditions where the a double-strand break in the desired DNA target which is generated by said variant leads to the activation of a positive selection marker or a reporter gene, or the inactivation of a negative selection marker or a reporter gene, by recombination-mediated repair of said DNA double-strand break, and recovering a subset of meganuclease variants able to cleave said desired DNA target;   f) isolating the genes encoding the meganuclease variants obtained in e) which are able to cleave said desired DNA target.   
     
     
         84 . The method according to  claim 83 , wherein said selection and screening steps use the phage display method. 
     
     
         85 . The method according to  claim 83 , wherein said amino acid residues that interact directly or indirectly with the DNA target of the initial meganuclease are selected from the group consisting of: D, E, H, K, N, Q, R, S, T and Y. 
     
     
         86 . The method according to  claim 85 , wherein said amino acid residues are substituted with an amino acid selected from the group consisting of: A, D, E, G, H, K, N, P, Q, S and T. 
     
     
         87 . The method according to  claim 83 , wherein said initial LAGLIDADG meganuclease is a natural or a modified meganuclease. 
     
     
         88 . The method according to  claim 83 , wherein said initial LAGLIDADG meganuclease is selected from the group consisting of: I-Cre I, I-Dmo I, PI-Sce I, and PI-Pfu I. 
     
     
         89 . The method according to  claim 88 , wherein said initial LAGLIDADG meganuclease is I-Cre I. 
     
     
         90 . The method according to  claim 89 , wherein I-Cre I variants are prepared by introducing amino acid substitutions in at least three positions of the I-CreI of SEQ ID NO: 10, wherein said positions are selected from the group consisting of Q26, K28, N30, S32, Y33, Q38, Q44, R68 and R70 of SEQ ID NO:10. 
     
     
         91 . The method according to  claim 90 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, N30, Y33, Q38, Q44, R68 and R70. 
     
     
         92 . The method according to  claim 90 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, N30, Y33, Q44, R68 and R70. 
     
     
         93 . The method according to  claim 90 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, Y33, Q38, Q44, R68 and R70. 
     
     
         94 . The method according to  claim 90 , further introducing an amino acid substitution with an uncharged amino acid, at position 75 of the I-Cre I of SEQ ID NO: 10. 
     
     
         95 . The method according to  claim 64 , wherein said uncharged amino acid is an asparagine residue. 
     
     
         96 . The method according to  claim 83 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprises an expression cassette comprising said desired DNA target and the coding region of an active negative selection marker, the coding region of an inactive positive selection marker, or the coding region of an active or inactive reporter gene product. 
     
     
         97 . The method according to  claim 83 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprises an expression cassette comprising (i) said desired DNA target and a polynucleotide comprising two direct repeats of an internal portion of a polynucleotide encoding an inactive positive selection marker, wherein the two repeats are separated by the desired DNA target, or (ii) said desired DNA target and a polynucleotide comprising two direct repeats of an internal portion of an inactive reporter gene, wherein the two repeats are separated by the desired DNA target. 
     
     
         98 . The method according to  claim 96 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprise an expression cassette comprising said desired DNA target and (i) a polynucleotide encoding an inactive positive selection marker due to the presence of a mutation or a deletion, or (ii) an inactive reporter gene due to the presence of a mutation or a deletion, wherein said desired DNA target is inserted in (i) or (ii) at the place of the deletion or in the vicinity of the mutation, and wherein the isolated yeast or mammalian cell further comprises a genetic construct comprising the segment of the polynucleotide encoding the positive selection marker which has been deleted or mutated, or the segment of the reporter gene which has been deleted or mutated, flanked at each side by the fragments of the polynucleotide encoding the positive selection marker or the reporter gene bordering the deletion/insertion. 
     
     
         99 . The method according to  claim 83 , wherein the double-strand break in the desired DNA target is repaired by homologous recombination between two direct repeats. 
     
     
         100 . The method according to  claim 83 , wherein the double-strand break in the desired DNA target is repaired by gene conversion. 
     
     
         101 . The method according to  claim 96 , wherein the expression cassette is inserted in a plasmid which is maintained in an episomal form in said isolated yeast or mammalian cell. 
     
     
         102 . The method according to  claim 96 , wherein the expression cassette is integrated in the chromosome of said isolated yeast or mammalian cell. 
     
     
         103 . The method according to  claim 83 , wherein said positive selection marker is an antibiotic resistance or an auxotrophy marker. 
     
     
         104 . The method according to  claim 96 , wherein said expression cassette comprises an additional selection marker. 
     
     
         105 . A method for producing LAGLIDADG meganuclease variants derived from an initial LAGLIDADG meganuclease, said meganuclease variants being able to cleave a desired DNA target which is different from the DNA target of the initial LAGLIDADG meganuclease, wherein said method comprises:
 a) generating a combinatorial library of genes coding for LAGLIDADG meganuclease variants by introducing diversity into several codons of a gene encoding said initial LAGLIDADG meganuclease, wherein said codons code for amino acid residues of the four-stranded beta-sheet of one domain or one subunit of said initial LAGLIDADG meganuclease that interact directly or indirectly with different bases of the DNA target of said initial meganuclease;   b) selecting meganuclease variants encoded by the genes of the combinatorial library that are able to bind said desired DNA target in vitro, under conditions excluding the presence of manganese or magnesium by incubating said library with said immobilized desired DNA target and recovering only the subset of bound meganuclease variants;   c) screening the subset of bound meganuclease variants obtained in b) by a binding assay consisting of incubation of said selected meganuclease variants with said desired DNA target immobilized on a support and recovering a subset of bound meganuclease variants;   d) screening the genes encoding the subset of bound meganuclease variants obtained in c) by an in vivo cleavage assay in an isolated yeast cell or an isolated mammalian cell, under conditions where the a double-strand break in the desired DNA target which is generated by said variant leads to the activation of a positive selection marker or a reporter gene, or the inactivation of a negative selection marker or a reporter gene, by recombination-mediated repair of said DNA double-strand break, and   e) isolating the genes encoding the meganuclease variants obtained in d) which are able to cleave said DNA target.   
     
     
         106 . The method according to  claim 105 , wherein said selection and screening steps use the phage display method. 
     
     
         107 . The method according to  claim 105 , wherein said amino acid residues that interact directly or indirectly with the DNA target of the initial meganuclease are selected from the group consisting of: D, E, H, K, N, Q, R, S, T and Y. 
     
     
         108 . The method according to  claim 107 , wherein said amino acid residues are substituted with an amino acid selected from the group consisting of: A, D, E, G, H, K, N, P, Q, S and T. 
     
     
         109 . The method according to  claim 105 , wherein said initial LAGLIDADG meganuclease is a natural or a modified meganuclease. 
     
     
         110 . The method according to  claim 105 , wherein said initial LAGLIDADG meganuclease is selected from the group consisting of: I-Cre I, I-Dmo I, PI-Sce I, and PI-Pfu I. 
     
     
         111 . The method according to  claim 110 , wherein said initial LAGLIDADG meganuclease is I-Cre I. 
     
     
         112 . The method according to  claim 111 , wherein I-Cre I variants are prepared by introducing amino acid substitutions in at least three positions of the I-CreI of SEQ ID NO: 10, wherein said positions are selected from the group consisting of Q26, K28, N30, S32, Y33, Q38, Q44, R68 and R70 of SEQ ID NO:10. 
     
     
         113 . The method according to  claim 112 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, N30, Y33, Q38, Q44, R68 and R70. 
     
     
         114 . The method according to  claim 112 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, N30, Y33, Q44, R68 and R70. 
     
     
         115 . The method according to  claim 112 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, Y33, Q38, Q44, R68 and R70. 
     
     
         116 . The method according to  claim 112 , further introducing an amino acid substitution with an uncharged amino acid, at position 75 of the I-Cre I of SEQ ID NO: 10. 
     
     
         117 . The method according to  claim 116 , wherein said uncharged amino acid is an asparagine residue. 
     
     
         118 . The method according to  claim 105 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprises an expression cassette comprising said desired DNA target and the coding region of an active negative selection marker, the coding region of an inactive positive selection marker, or the coding region of an active or inactive reporter gene product. 
     
     
         119 . The method according to  claim 105 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprises an expression cassette comprising (i) said desired DNA target and a polynucleotide comprising two direct repeats of an internal portion of a polynucleotide encoding an inactive positive selection marker, wherein the two repeats are separated by the desired DNA target, or (ii) said desired DNA target and a polynucleotide comprising two direct repeats of an internal portion of an inactive reporter gene, wherein the two repeats are separated by the desired DNA target. 
     
     
         120 . The method according to  claim 118 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprise an expression cassette comprising said desired DNA target and (i) a polynucleotide encoding an inactive positive selection marker due to the presence of a mutation or a deletion, or (ii) an inactive reporter gene due to the presence of a mutation or a deletion, wherein said desired DNA target is inserted in (i) or (ii) at the place of the deletion or in the vicinity of the mutation, and wherein the isolated yeast or mammalian cell further comprises a genetic construct comprising the segment of the polynucleotide encoding the positive selection marker which has been deleted or mutated, or the segment of the reporter gene which has been deleted or mutated, flanked at each side by the fragments of the polynucleotide encoding the positive selection marker or the reporter gene bordering the deletion/insertion. 
     
     
         121 . The method according to  claim 105 , wherein the double-strand break in the desired DNA target is repaired by homologous recombination between two direct repeats. 
     
     
         122 . The method according to  claim 105 , wherein the double-strand break in the desired DNA target is repaired by gene conversion. 
     
     
         123 . The method according to  claim 118 , wherein the expression cassette is inserted in a plasmid which is maintained in an episomal form in said isolated yeast or mammalian cell. 
     
     
         124 . The method according to  claim 118 , wherein the expression cassette is integrated in the chromosome of said isolated yeast or mammalian cell. 
     
     
         125 . The method according to  claim 105 , wherein said positive selection marker is an antibiotic resistance or an auxotrophy marker. 
     
     
         126 . The method according to  claim 118 , wherein said expression cassette comprises an additional selection marker. 
     
     
         127 . A method for producing LAGLIDADG meganuclease variants derived from an initial LAGLIDADG meganuclease, said meganuclease variants being able to cleave a desired DNA target which is different from the DNA target of the initial LAGLIDADG meganuclease, wherein said method comprises:
 a) generating a combinatorial library of genes coding for LAGLIDADG meganuclease variants by introducing diversity into several codons of a gene encoding said initial LAGLIDADG meganuclease, wherein said codons code for amino acid residues of the four-stranded beta-sheet of one domain or one subunit of said initial LAGLIDAG meganuclease that interact directly or indirectly with different bases of the DNA target of said initial meganuclease;   b) selecting meganuclease variants encoded by the genes of the combinatorial library that are able to bind said desired DNA target in vitro, under conditions excluding the presence of manganese or magnesium by incubating said library with said immobilized desired DNA target and recovering only the subset of bound meganuclease variants;   c) selecting among said subset of bound meganuclease variants obtained in b), the meganuclease variants able to cleave said desired DNA target, by an in vivo cleavage assay, in an isolated yeast cell or an isolated mammalian cell, under conditions where the double-strand break in the desired DNA target which is generated by said variant leads to the activation of a positive selection marker, or the inactivation of a negative selection marker, by recombination-mediated repair of said DNA double-strand break, and recovering the subset of meganuclease variants able to cleave said desired DNA target;   d) screening said subset of meganuclease variants obtained in step c) by an in vivo cleavage assay in an isolated yeast cell or an isolated mammalian cell, under conditions where a double-strand break in the desired DNA target which is generated by said variant leads to the activation of a positive selection marker or a reporter gene, or the inactivation of a negative selection marker or a reporter gene, by recombination-mediated repair of said DNA double-strand break, and   e) isolating the meganuclease variants obtained in d) which are able to cleave said desired DNA target.   
     
     
         128 . The method according to  claim 127 , wherein said selection and screening steps use the phage display method. 
     
     
         129 . The method according to  claim 127 , wherein said amino acid residues that interact directly or indirectly with the DNA target of the initial meganuclease are selected from the group consisting of: D, E, H, K, N, Q, R, S, T and Y. 
     
     
         130 . The method according to  claim 129 , wherein said amino acid residues are substituted with an amino acid selected from the group consisting of: A, D, E, G, H, K, N, P, Q, S and T. 
     
     
         131 . The method according to  claim 127 , wherein said initial LAGLIDADG meganuclease is a natural or a modified meganuclease. 
     
     
         132 . The method according to  claim 127 , wherein said initial LAGLIDADG meganuclease is selected from the group consisting of: I-Cre I, I-Dmo I, PI-Sce I, and PI-Pfu I. 
     
     
         133 . The method according to  claim 132 , wherein said initial LAGLIDADG meganuclease is I-Cre I. 
     
     
         134 . The method according to  claim 133 , wherein I-Cre I variants are prepared by introducing amino acid substitutions in at least three positions of the I-CreI of SEQ ID NO: 10, wherein said positions are selected from the group consisting of Q26, K28, N30, S32, Y33, Q38, Q44, R68 and R70 of SEQ ID NO:10. 
     
     
         135 . The method according to  claim 134 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, N30, Y33, Q38, Q44, R68 and R70. 
     
     
         136 . The method according to  claim 134 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, N30, Y33, Q44, R68 and R70. 
     
     
         137 . The method according to  claim 134 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, Y33, Q38, Q44, R68 and R70. 
     
     
         138 . The method according to  claim 134 , further introducing an amino acid substitution with an uncharged amino acid, at position 75 of the I-Cre I of SEQ ID NO: 10. 
     
     
         139 . The method according to  claim 138 , wherein said uncharged amino acid is an asparagine residue. 
     
     
         140 . The method according to  claim 127 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprises an expression cassette comprising said desired DNA target and the coding region of an active negative selection marker, the coding region of an inactive positive selection marker, or the coding region of an active or inactive reporter gene. 
     
     
         141 . The method according to  claim 127 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprises an expression cassette comprising (i) said desired DNA target and a polynucleotide comprising two direct repeats of an internal portion of a polynucleotide encoding an inactive positive selection marker, wherein the two repeats are separated by the desired DNA target, or (ii) said desired DNA target and a polynucleotide comprising two direct repeats of an internal portion of an inactive reporter gene, wherein the two repeats are separated by the desired DNA target. 
     
     
         142 . The method according to  claim 140 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprise an expression cassette comprising said desired DNA target and (i) a polynucleotide encoding an inactive positive selection marker due to the presence of a mutation or a deletion, or (ii) an inactive reporter gene due to the presence of a mutation or a deletion, wherein said desired DNA target is inserted in (i) or (ii) at the place of the deletion or in the vicinity of the mutation, and wherein the isolated yeast or mammalian cell further comprises a genetic construct comprising the segment of the polynucleotide encoding the positive selection marker which has been deleted or mutated, or the segment of the reporter gene which has been deleted or mutated, flanked at each side by the fragments of the polynucleotide encoding the positive selection marker or the reporter gene bordering the deletion/insertion. 
     
     
         143 . The method according to  claim 127 , wherein the double-strand break in the desired DNA target is repaired by homologous recombination between two direct repeats. 
     
     
         144 . The method according to  claim 127 , wherein the double-strand break in the desired DNA target is repaired by gene conversion. 
     
     
         145 . The method according to  claim 140 , wherein the expression cassette is inserted in a plasmid which is maintained in an episomal form in said isolated yeast or mammalian cell. 
     
     
         146 . The method according to  claim 114 , wherein the expression cassette is integrated in the chromosome of said isolated yeast or mammalian cell. 
     
     
         147 . The method according to  claim 127 , wherein said positive selection marker is an antibiotic resistance or an auxotrophy marker. 
     
     
         148 . The method according to  claim 140 , wherein said expression cassette comprises an additional selection marker. 
     
     
         149 . A method for producing LAGLIDADG meganuclease variants derived from an initial LAGLIDADG meganuclease, said meganuclease variants being able to cleave a desired DNA target which is different from the DNA target of the initial LAGLIDADG meganuclease, wherein said method comprises:
 a) generating a combinatorial library of genes coding for LAGLIDADG meganuclease variants by introducing diversity into several codons of a gene encoding said initial LAGLIDADG meganuclease, wherein said codons code for amino acid residues of the four-stranded beta-sheet of one domain or one subunit of said initial LAGLIDADG meganuclease that interact directly or indirectly with different bases of the DNA target of said initial meganuclease;   b) screening said meganuclease variants encoded by the genes of the combinatorial library obtained in a) by a binding assay consisting of incubating said meganuclease variants encoded by the genes of the combinatorial library with said desired DNA target immobilized on a support and recovering a subset of meganuclease variants that are bound to the desired DNA target;   c) screening said subset of meganuclease variants obtained in b) by an in vivo cleavage assay in an isolated yeast cell or an isolated mammalian cell, under conditions where the double-strand break in the desired DNA target which is generated by said variant leads to the activation of a positive selection marker or a reporter gene, or the inactivation of a negative selection marker or a reporter gene, by recombination-mediated repair of said DNA double-strand break, and   d) isolating the meganuclease variants obtained in c) which are able to cleave said desired DNA target.   
     
     
         150 . The method according to  claim 149 , wherein said selection and screening steps use the phage display method. 
     
     
         151 . The method according to  claim 149 , wherein said amino acid residues that interact directly or indirectly with the DNA target of the initial meganuclease are selected from the group consisting of: D, E, H, K, N, Q, R, S, T and Y. 
     
     
         152 . The method according to  claim 149 , wherein said amino acid residues are substituted with an amino acid selected from the group consisting of: A, D, E, G, H, K, N, P, Q, S and T. 
     
     
         153 . The method according to  claim 149 , wherein said initial LAGLIDADG meganuclease is a natural or a modified meganuclease. 
     
     
         154 . The method according to  claim 149 , wherein said initial LAGLIDADG meganuclease is selected from the group consisting of: I-Cre I, I-Dmo I, PI-Sce I, and PI-Pfu I. 
     
     
         155 . The method according to  claim 154 , wherein said initial LAGLIDADG meganuclease is I-Cre I. 
     
     
         156 . The method according to  claim 155 , wherein I-Cre I variants are prepared by introducing amino acid substitutions in at least three positions of the I-CreI of SEQ ID NO: 10, wherein said positions are selected from the group consisting of Q26, K28, N30, S32, Y33, Q38, Q44, R68 and R70 of SEQ ID NO:10. 
     
     
         157 . The method according to  claim 156 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, N30, Y33, Q38, Q44, R68 and R70. 
     
     
         158 . The method according to  claim 156 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, N30, Y33, Q44, R68 and R70. 
     
     
         159 . The method according to  claim 156 , wherein said I-Cre I variants comprise amino acid substitutions at the following positions Q26, K28, Y33, Q38, Q44, R68 and R70. 
     
     
         160 . The method according to  claim 156 , further introducing an amino acid substitution with an uncharged amino acid, at position 75 of the I-Cre I of SEQ ID NO: 10. 
     
     
         161 . The method according to  claim 160 , wherein said uncharged amino acid is an asparagine residue. 
     
     
         162 . The method according to  claim 149 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprises an expression cassette comprising said desired DNA target and the coding region of an active negative selection marker, the coding region of an inactive positive selection marker, or the coding region of an active or inactive reporter gene. 
     
     
         163 . The method according to  claim 149 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprises an expression cassette comprising (i) said desired DNA target and a polynucleotide comprising two direct repeats of an internal portion of a polynucleotide encoding an inactive positive selection marker, wherein the two repeats are separated by the desired DNA target, or (ii) said desired DNA target and a polynucleotide comprising two direct repeats of an internal portion of an inactive reporter gene, wherein the two repeats are separated by the desired DNA target. 
     
     
         164 . The method according to  claim 162 , wherein in said in vivo cleavage assay, said isolated yeast or mammalian cell comprise an expression cassette comprising said desired DNA target and (i) a polynucleotide encoding an inactive positive selection marker due to the presence of a mutation or a deletion, or (ii) an inactive reporter gene due to the presence of a mutation or a deletion, wherein said desired DNA target is inserted in (i) or (ii) at the place of the deletion or in the vicinity of the mutation, and wherein the isolated yeast or mammalian cell further comprises a genetic construct comprising the segment of the polynucleotide encoding the positive selection marker which has been deleted or mutated, or the segment of the reporter gene which has been deleted or mutated, flanked at each side by the fragments of the polynucleotide encoding the positive selection marker or the reporter gene bordering the deletion/insertion. 
     
     
         165 . The method according to  claim 149 , wherein the double-strand break in the desired DNA target is repaired by homologous recombination between two direct repeats. 
     
     
         166 . The method according to  claim 149 , wherein the double-strand break in the desired DNA target is repaired by gene conversion. 
     
     
         167 . The method according to  claim 162 , wherein the expression cassette is inserted in a plasmid which is maintained in an episomal form in said isolated yeast or mammalian cell. 
     
     
         168 . The method according to  claim 162 , wherein the expression cassette is integrated in the chromosome of said isolated yeast or mammalian cell. 
     
     
         169 . The method according to  claim 149 , wherein said positive selection marker is an antibiotic resistance or an auxotrophy marker. 
     
     
         170 . The method according to  claim 162 , wherein said expression cassette comprises an additional selection marker.