Tevi chimeric endonuclease and their preferential cleavage sites

Abstract

The present invention relates to a method to cleave target nucleic acid sequence by the catalytic domain of a GIY-YIG homing endonucleases 1-Tevl. More precisely, the invention relates to the deciphering of new preferential 1-Tevl cleavage sites for efficient and specific cleavage activity. The invention concerns a method for the generation of TevI specific chimeric endonucleases to target nucleic acid sequence including such cleavage sites and methods of using same for gene editing.

1 . A method for generating a TevI chimeric endonuclease comprising the steps of:
 a. determining a target nucleic acid sequence comprising a I-TevI cleavage site selected from the group consisting of: SEQ ID NO: 2 to SEQ ID NO 117;   b. selecting or engineering at least one nucleic acid binding domain to specifically bind a recognition site adjacent to said cleavage site;   c. fusing said nucleic acid binding domain with at least one catalytic domain of I-TevI.   
     
     
         2 . The method according to  claim 1 , wherein said cleavage site is selected from the group consisting of: SEQ ID NO: 2 to 55. 
     
     
         3 . The method according to  claim 1 , wherein said I-TevI catalytic domain protein sequence is a part of the protein sequence SEQ ID NO: 257 
     
     
         4 . The method of  claim 3 , wherein said I-TevI catalytic domain protein sequence is selected from the group consisting of: SEQ ID NO: 268 and SEQ ID NO: 284. 
     
     
         5 . The method of  claim 4 , wherein said I-TevI catalytic domain protein sequence shares at least 80% protein sequence identity with a protein sequence selected from the group consisting of: SEQ ID NO: 268 and SEQ ID NO: 284. 
     
     
         6 . The method according to  claim 1 , wherein said DNA nucleic acid binding domain is an engineered MBBBD binding domain. 
     
     
         7 . The method according to  claim 1 , wherein said DNA binding domain is an engineered TALE binding domain comprising a plurality of TALE like repeat sequences, each repeat comprising a RVD specific to each nucleotide base of a TALE DNA binding site. 
     
     
         8 . The method according to  claim 1 , wherein said I-TevI catalytic domain is fused to the N-terminal part of said nucleic acid binding domain. 
     
     
         9 . The method according to  claim 1 , wherein said I-TevI catalytic domain is fused to the C-terminal part of said nucleic acid binding domain. 
     
     
         10 . The method according to  claim 1 , wherein said I-TevI catalytic domain is fused to said nucleic acid binding domain by a peptide linker. 
     
     
         11 . The method according to  claim 1 , wherein said TevI chimeric endonuclease is selected from the group consisting of: SEQ ID NO: 452 to 458. 
     
     
         12 . The method according to  claim 1 , wherein said target nucleic acid sequence comprises said recognition site spaced away from said I-TevI cleavage site by an optimal distance to increase cleavage activity. 
     
     
         13 . The method according to  claim 12 , wherein T 0  of said TALE nucleic acid recognition site is located between 5 to 20 bp, preferably between 6 to 12 bp, more preferably 10 bp away from terminal G base of said I-TevI cleavage site. 
     
     
         14 . The method according to  claim 1 , wherein said target nucleic acid sequence comprises a nucleotide A or T after the terminal G base of said I-TevI cleavage site. 
     
     
         15 . A TevI chimeric endonuclease obtained from the method according to  claim 1 . 
     
     
         16 . A TevI chimeric endonuclease having at least 80% amino acid sequence identity with a protein sequence selected from the group consisting of: SEQ ID NO: 452 to 458. 
     
     
         17 . A method to selectively cleave a target nucleic acid by using catalytic domain of I-TevI, said method comprising:
 a. selecting a target nucleic acid sequence comprising a cleavage site selected from the group consisting of: SEQ ID NO: 2 to SEQ ID NO 117, preferably SEQ ID NO: 2 to 55;   b. selecting or engineering a nucleic acid binding domain to specifically bind a recognition site adjacent to said cleavage site;   c. fusing said nucleic acid binding domain with said catalytic domain of I-TevI to obtain a TevI chimeric endonuclease;   d. contacting said target nucleic acid with said chimeric endonuclease.   
     
     
         18 . The method according to  claim 17 , further comprising a step of introducing said cleavage site selected in step a) into said target nucleic acid in order to be cleaved by said TevI chimeric endonuclease. 
     
     
         19 . A method to process a target nucleic acid sequence in a cell comprising:
 a. generating a TevI chimeric endonuclease according to  claim 1 ;   b. introducing said TevI chimeric endonuclease into said cell.   
     
     
         20 . The method according to  claim 19 , further comprising providing to the cell an exogeneous nucleic acid comprising a sequence homologous to at least a portion of the target nucleic acid sequence, such that homologous recombination occurs between the target DNA sequence and the exogeneous nucleic acid. 
     
     
         21 . The method according to  claim 19 , to increase mutagenesis, further comprising a step of providing and contacting an additional catalytic domain with the target nucleic acid sequence. 
     
     
         22 . The method of  claim 21 , wherein said catalytic domain is a DNA end-processing enzyme. 
     
     
         23 . The method of  claim 22  wherein said DNA end-processing enzyme is Trex2. 
     
     
         24 . The method of  claim 23  wherein said DNA processing enzyme is a single chain Trex2. 
     
     
         25 . The method according to  claim 19 , wherein said cell is an animal or plant cell. 
     
     
         26 . The method according to  claim 25 , wherein the cell is a plant cell and further comprising developing the plant cell into a whole plant. 
     
     
         27 . The method according to  claim 25 , wherein said animal cell is a progenitor cell and further comprising obtaining a transgenic animal from said progenitor cell.