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 a meganuclease variant derived from an initial meganuclease, said meganuclease variant being able to cleave a DNA target sequence which is different from the recognition and cleavage site of the initial meganuclease, characterized in that it comprises the following steps: 
 a) preparing a library of meganuclease variants from an initial meganuclease and,    b) selecting and/or screening the variants able to cleave said DNA target sequence, in vivo, under conditions where the double-strand break in the DNA target sequence 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.    
     
     
         42 . The method according to  claim 41 , characterized in that step b) comprises the use of a cell modified by at least an expression vector comprising said DNA target sequence and the coding sequence for a negative selection marker in an active form, a positive selection marker in an inactive form, or a reporter gene in an active or inactive form.  
     
     
         43 . The method according to  claim 42 , characterized in that said cell is modified by an expression vector comprising a modified positive selection marker or reporter gene, said selection marker or reporter gene comprising an internal duplication separated by an intervening sequence including the target DNA sequence, and eventually an additional selection marker.  
     
     
         44 . The method according to  claim 42 , characterized in that said cell is modified by an expression vector comprising a first modified positive selection marker or reporter gene, said selection marker or reporter gene comprising a mutation or a deletion and an insertion of the target DNA at the place of the deletion or in the vicinity of the mutation, said cell being further modified by the segment of the positive selection marker or reporter gene which has been deleted or mutated, flanked at each side by the positive selection marker or reporter gene sequences bordering the deletion/insertion.  
     
     
         45 . The method according to  claim 41 , characterized in that the double-strand break in the DNA target sequence is repaired by homologous recombination between two direct repeats.  
     
     
         46 . The method according to  claim 41 , characterized in that the double-strand break in the DNA target sequence is repaired by gene conversion.  
     
     
         47 . The method according to  claim 42 , characterized in that the coding sequence and the DNA target sequence are on a plasmid.  
     
     
         48 . The method according to  claim 42 , characterized in that the coding sequence and the DNA target sequence are integrated in the chromosome of said cell.  
     
     
         49 . The method according to  claim 41 , characterized in that said positive selection marker is an antibiotic resistance or an auxotrophy marker.  
     
     
         50 . The method according to  claim 42 , characterized in that said cell is a yeast cell.  
     
     
         51 . The method according to  claim 42 , characterized in that said cell is a mammalian cell.  
     
     
         52 . The method according to  claim 41 , characterized in that it consists in step a) and a step b) which is a selection step for the cleavage activity.  
     
     
         53 . The method according to  claim 41 , characterized in that it consists in step a) and a step b) which is a screening step for the cleavage activity.  
     
     
         54 . The method according to  claim 41 , characterized in that it consists in step a) and step b) which is a combination of a selection step for the cleavage activity and a screening step for the cleavage activity.  
     
     
         55 . The method according to  claim 41 , characterized in that it consists in step a) and a step b) which is included in a combination of selection and screening steps selected from the group consisting of: 
 a selection step for the binding ability, a screening step for the binding ability, a selection step for the cleavage activity, and a screening step for the cleavage activity;    a selection step for the binding ability, a screening step for the binding ability, and a screening step for the cleavage activity;    a selection step for the binding ability, a selection step for the cleavage activity, and a screening step for the cleavage activity; or,    a screening step for the binding ability and a screening step for the cleavage activity.    
     
     
         56 . The method according to  claim 55 , characterized in that said selection and screening steps for the binding ability use the phage display.  
     
     
         57 . The method according to  claim 52 , characterized in that said selection for the cleavage activity uses a test in which the cleavage leads to the activation of a positive selection marker.  
     
     
         58 . The method according to  claim 53 , characterized in that and said screening for the cleavage activity uses a test in which the cleavage leads to the activation of a reporter gene.  
     
     
         59 . The method according to  claim 41 , characterized in that said meganuclease variants have amino acid variations at positions contacting the DNA target or interacting directly or indirectly with said DNA target.  
     
     
         60 . The method according to  claim 59 , characterized in that said amino acid variations are replacement of the initial amino acid with an amino acid selected from the group consisting of: D, E, H, K, N, Q, R, S, T, Y.  
     
     
         61 . The method according to  claim 41  characterized in that said initial meganuclease is a natural or a modified meganuclease.  
     
     
         62 . The method according to  claim 61  characterized in that said initial meganuclease is a homing endonuclease.  
     
     
         63 . The method according to  claim 62  characterized in that said homing endonuclease is a LAGLIDADG homing endonuclease.  
     
     
         64 . The method according to  claim 63 , characterized in that said LAGLIDADG homing endonuclease is selected from the group consisting of: I-Cre I, I-Dmo I, PI-Sce I, and PI-Pfu I.  
     
     
         65 . The method according to  claim 64 , characterized in that said LAGLIDADG homing endonuclease is I-Cre I.  
     
     
         66 . The method according to  claim 61 , characterized in that said initial meganuclease is a hybrid meganuclease.  
     
     
         67 . The method according to  claim 66 , characterized in that said hybrid meganuclease is the hybrid homing endonuclease I-Dmo I/I Cre I.  
     
     
         68 . The method according to  claim 65 , characterized in that I-Cre I variants are prepared by introducing amino acid diversity in positions selected from the group consisting of: Q26, K28, N30, S32, Y33, Q38, Q44, R68, R70 and T140.  
     
     
         69 . The method according to  claim 68 , characterized in that said I-Cre I variants are prepared by introducing amino acid diversity in positions: a) Q26, K28, N30, Y33, Q38, Q44, R68, R70, T140; b) Q26, K28, N30, Y33, Q38, Q44, R68, R70; c) Q26, K28, N30, Y33, Q44, R68, R70; or d) Q26, K28, Y33, Q38, Q44, R68, R70.  
     
     
         70 . The method according to  claim 69 , characterized in that said I-Cre I variants are prepared by introducing amino acid diversity in positions: Q26, K28, N30, Y33, Q38, Q44, R68 and R70.  
     
     
         71 . The method according to  claim 65 , characterized in that the I-Cre I or I-Dmo I/I Cre I variants further comprise the mutation of the aspartic acid in position 75 of I-Cre I, in an uncharged amino acid.  
     
     
         72 . The method according to  claim 71 , characterized in that said uncharged amino acid is an asparagine residue.