Modular base-specific nucleic acid binding domains from burkholderia rhizoxinica proteins

Abstract

The present invention concerns new modular base-per-base specific nucleic acid binding domains (MBBBD) derived from newly identified proteins from the bacterial endosymbiont Burkholderia Rhizoxinica and their use for engineering nucleic acid processing enzymes, such as specific endonucleases or transcription activators.

1 . A nucleic acid binding polypeptide that comprises a succession of modules from 30 to 35 amino acids, said succession of modules displaying a base per base specificity towards a nucleic acid target sequence, wherein at least one of said modules has at least 70% sequence identity with one of the module polypeptide sequences from the proteins E5AV36, E5AW43 and E5AW45 of  Burkholderia rhizoxinica.    
     
     
         2 . A nucleic acid binding polypeptide according to  claim 1 , wherein said module polypeptide sequence has at least 90% sequence identity with a polypeptide sequence selected from the group consisting of: SEQ ID NO: 162 to 181, SEQ ID NO: 31 to SEQ ID NO: 36 and SEQ ID NO: 37 to SEQ ID NO: 63. 
     
     
         3 . A nucleic acid binding polypeptide according to  claim 1  wherein at least one of said modules coming from the proteins E5AV36, E5AW43, and E5AW45, comprise a unique variable amino acid residue that determines the specificity of each module towards a nucleotide base. 
     
     
         4 . A nucleic acid binding polypeptide according to  claim 3 , wherein said unique variable residue determines the following nucleic acid base: 
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   AA 
                   Nucleotide base 
                 
                     
                     
                 
                     
                   I, S, T 
                   A 
                 
                     
                   G, R 
                   T 
                 
                     
                   D, T, * 
                   C 
                 
                     
                   N, R 
                   G 
                 
                     
                     
                 
             
                
                
                
               
               
                
                
                
                
                
               
            
           
         
       
     
     
         5 . A nucleic acid binding polypeptide according to  claim 4 , wherein said variable residue is located in position 13 of each module. 
     
     
         6 . A nucleic acid polypeptide according to  claim 1  wherein at least one of said modules coming from the proteins E5AV36, E5AW43, and E5AW45, comprise(s) variable residues in position 12 and 13 NT, **, KG, NR, HH, HS, RN, which determine the specificity of said module towards a nucleotide base. 
     
     
         7 . A nucleic acid binding polypeptide according to  claim 1  wherein said target nucleic acid sequence does not comprise a thymidine nucleotide at position −1. 
     
     
         8 . A nucleic acid binding polypeptide according to  claim 1 , wherein said polypeptide further comprises a module that has at least 80% amino acid identity with an AvrBs3 repeat of SEQ ID NO. 10. 
     
     
         9 . A polypeptide comprising a nucleic acid binding polypeptide according to  claim 1 , wherein said nucleic acid binding polypeptide is fused to a catalytic domain. 
     
     
         10 . The polypeptide according to  claim 9 , wherein said catalytic domain has an activity of transcriptional activation or inhibition. 
     
     
         11 . The polypeptide according to  claim 9 , wherein said catalytic domain has a nuclease activity. 
     
     
         12 . The polypeptide according to  claim 11 , wherein said catalytic domain has an endonuclease activity. 
     
     
         13 . The polypeptide according to  claim 12 , wherein said polypeptide has cleavage activity under dimeric form. 
     
     
         14 . The polypeptide according to  claim 13 , wherein said catalytic domain comes from the endonuclease FokI. 
     
     
         15 . The polypeptide according to  claim 12 , wherein said polypeptide has cleavage activity under monomeric form. 
     
     
         16 . The polypeptide according to  claim 15 , wherein said catalytic domain comes from the endonuclease I-TevI. 
     
     
         17 . The polypeptide according to  claim 9 , wherein said catalytic domain is from a homing endonuclease. 
     
     
         18 . The polypeptide according to  claim 9 , for use to process the genetic material of a cell within or adjacent to said nucleic acid target sequence. 
     
     
         19 . The polypeptide according to  claim 1  further comprising a polypeptide sequence having at least 80% amino acid identity with the C-terminal or N-terminal polypeptide from AvrBs3 (SEQ ID NO.6 and SEQ ID NO.111). 
     
     
         20 . The polypeptide according to  claim 1  for use as a therapeutic molecule. 
     
     
         21 . A polynucleotide, DNA or RNA vector, plasmid or virus encoding a polypeptide according to  claim 1 . 
     
     
         22 . A method for targeting the genetic material of a cell, comprising:
 (a) providing a cell comprising a nucleic acid target sequence; and   (b) introducing into the cell a polypeptide according to  claim 9 ;   such that said polypeptide processes the genetic material of the cell within or adjacent to said target nucleic acid sequence.   
     
     
         23 . The method for modifying the genetic material of a cell according to  claim 22 , wherein said polypeptide cleaves genetic material within or adjacent to said target nucleic acid sequence. 
     
     
         24 . The method according to  claim 23 , further comprising introducing into the cell an exogenous nucleic acid comprising a sequence homologous to at least a portion of the target nucleic acid sequence, such that homologous recombination occurs between said exogenous nucleic acid and the target nucleic acid sequence. 
     
     
         25 . The method according to  claim 22 , wherein the cell is a plant cell. 
     
     
         26 . The method according to  claim 22 , wherein the cell is a mammalian cell. 
     
     
         27 . A method for generating an animal comprising:
 (a) providing a eukaryotic cell comprising a nucleic acid target sequence into which it is desired to introduce a genetic modification;   (b) generating a cleavage within or adjacent to said target nucleic acid sequence by introducing a polypeptide according to  claim 11  into said cell; and   (c) generating an animal from the cell or progeny thereof, in which a cleavage has occurred.   
     
     
         28 . A method according to  claim 27 , further comprising: introducing into the cell an exogenous nucleic acid comprising a sequence homologous to at least a portion of the nucleic acid target sequence, such that homologous recombination occurs between said exogenous nucleic acid and the nucleic acid target sequence in the cell or progeny thereof; and generating an animal from the cell or progeny thereof in which homologous recombination has occurred. 
     
     
         29 . A method for generating a plant comprising:
 (a) providing a plant cell comprising a nucleic acid target sequence into which it is desired to introduce a genetic modification;   (b) generating a cleavage within or adjacent to said nucleic acid target sequence by introducing a polypeptide according to  claim 11 ; and   (c) generating a plant from the cell or progeny thereof in which a cleavage has occurred.   
     
     
         30 . The method according to  claim 29 , further comprising: introducing into the plant cell an exogenous nucleic acid comprising a sequence homologous to at least a portion of the target nucleic acid sequence, such that homologous recombination occurs between said exogenous nucleic acid and the target nucleic acid sequence in the cell or progeny thereof; and generating a plant from the cell or progeny thereof in which homologous recombination has occurred.