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 monomer of an I-CreI meganuclease variant comprising at least one mutation in the amino acid sequence of SEQ ID NO: 10, wherein said at least one mutation comprises a substitution at one or more of the amino acid residues at positions Q44, R68 and R70 and wherein said monomer when in a dimeric form has a modified DNA cleavage specificity relative to SEQ ID NO: 10. 
     
     
         42 . The monomer of an I-CreI variant of  claim 41  wherein said monomer comprises at least two mutations at positions 44, 68 and 70. 
     
     
         43 . The monomer of an I-CreI variant of  claim 41  wherein said monomer comprises mutations at positions 44, 68 and 70. 
     
     
         44 . An I-CreI meganuclease variant homodimer, wherein said homodimer comprises two identical monomers according to  claim 41 . 
     
     
         45 . An I-Crel meganuclease variant heterodimer, wherein said heterodimer comprises at least one monomer according to  claim 41  and a second monomer selected from the group consisting of a different monomer according to  claim 41 , a wild-type monomer from I-Crel, and a variant of the wild-type monomer from I-Crel. 
     
     
         46 . The I-Crel meganuclease variant heterodimer according to  claim 45 , wherein said heterodimer comprises two different monomers according to  claim 41 . 
     
     
         47 . The I-Crel meganuclease variant heterodimer according to  claim 45 , wherein said heterodimer comprises one monomer according to  claim 41  and a wild-type monomer from I-Crel. 
     
     
         48 . The I-Crel meganuclease variant heterodimer according to  claim 45 , wherein said heterodimer comprises one monomer according to  claim 41  and a variant of the wild-type monomer from I-Crel. 
     
     
         49 . A single-chain chimeric meganuclease comprising the fusion of at least one monomer according to  claim 41  and a second monomer selected from the group consisting of a different monomer according to  claim 41 , a wild-type monomer from I-Crel, and a variant of the wild-type monomer from I-Crel. 
     
     
         50 . The single-chain chimeric meganuclease according to  claim 49 , wherein said chimeric meganuclease comprises two different monomers according to  claim 41 . 
     
     
         51 . The single-chain chimeric meganuclease according to  claim 49 , wherein said chimeric meganuclease comprises one monomer according to  claim 41  and a wild-type monomer from I-Crel. 
     
     
         52 . The single-chain chimeric meganuclease according to  claim 49 , wherein said chimeric meganuclease comprises one monomer according to  claim 41  and a variant of the wild-type monomer from I-Crel. 
     
     
         53 . The monomer I-Crel meganuclease variant of  claim 41  wherein said monomer further comprises a mutation at D75. 
     
     
         54 . The single-chain chimeric meganuclease of  claim 49  wherein the monomer of claim  1  further comprises a mutation at D75. 
     
     
         55 . A polynucleotide fragment encoding a I-Crel variant according to  claim 41  or a single-chain chimeric endonuclease of  claim 49 . 
     
     
         56 . A recombinant vector comprising at least one polynucleotide fragment according to  claim 55 . 
     
     
         57 . The vector according to  claim 56 , which includes a targeting construct comprising sequences sharing homologies with a region surrounding a DNA target sequence. 
     
     
         58 . A host cell which is modified by a polynucleotide according to  claim 55  or a recombinant vector according to  claim 56 . 
     
     
         59 . A non-human transgenic animal which is modified by a polynucleotide according to  claim 55  or a recombinant vector according to  claim 56 . 
     
     
         60 . A transgenic plant which is modified by a polynucleotide according to  claim 55  or a recombinant vector according to  claim 56 .