Protein having nuclease activity, fusion proteins and uses thereof

Abstract

The present invention relates to a nucleic acid molecule encoding (I) a polypeptide having the activity of an endonuclease, which is (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1; (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 2; (c) a nucleic acid molecule encoding an endonuclease, the amino acid sequence of which is at least 70% identical to the amino acid sequence of SEQ ID NO: 1; (d) a nucleic acid molecule comprising or consisting of a nucleotide sequence which is at least 50% identical to the nucleotide sequence of SEQ ID NO: 2; (e) a nucleic acid molecule which is degenerate with respect to the nucleic acid molecule of (d); or (f) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (e) wherein T is replaced by U; (II) a fragment of the polypeptide of (I) having the activity of an endonuclease. Also, the present invention relates to a vector comprising the nucleic acid molecule and a protein encoded by said nucleic acid molecule. Further, the invention relates to a method of modifying the genome of a eukaryotic cell and a method of producing a non-human vertebrate or mammal.

1 . A nucleic acid molecule encoding
 (I) a polypeptide having the activity of an endonuclease, which is selected from the group consisting of:
 (a) a nucleic acid molecule encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1; 
 (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 2; 
 (c) a nucleic acid molecule encoding an endonuclease, the amino acid sequence of which is at least 70% identical to the amino acid sequence of SEQ ID NO: 1; 
 (d) a nucleic acid molecule comprising or consisting of a nucleotide sequence which is at least 50% identical to the nucleotide sequence of SEQ ID NO: 2; 
 (e) a nucleic acid molecule which is degenerate with respect to the nucleic acid molecule of (d); and 
 (f) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (e) wherein T is replaced by U; or 
   (II) a fragment of the polypeptide of (I) having the activity of an endonuclease.   
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein in (I)(c) in said amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 the amino acid residues P66, D67, D84 and/or K86 of SEQ ID NO: 1 are not modified. 
     
     
         3 . The nucleic acid molecule of  claim 1  further encoding a DNA-binding domain. 
     
     
         4 . The nucleic acid molecule of  claim 3 , wherein the DNA-binding domain is a TAL effector motif of a TAL effector protein. 
     
     
         5 . A vector comprising the nucleic acid molecule of  claim 1 . 
     
     
         6 . A host cell comprising the nucleic acid molecule of  claim 1 . 
     
     
         7 . A protein or fusion protein having the activity of an endonuclease encoded by the nucleic acid molecule of  claim 1 . 
     
     
         8 . A method of modifying a target sequence in the genome of a eukaryotic cell, the method comprising the step of:
 (a) introducing into said cell the nucleic acid molecule of  claim 1 , a vector of comprising the nucleic acid molecule of  claim 1  or a protein or fusion protein having the activity of an endonuclease encoded by the nucleic acid molecule of  claim 1 .   
     
     
         9 . The method of  claim 8 , wherein the modification of said target sequence is by homologous recombination with a donor nucleic acid sequence, further comprising the step:
 (b) introducing a nucleic acid molecule into said cell, wherein said nucleic acid molecule comprises said donor nucleic acid sequence, wherein said donor DNA sequence is flanked upstream by a first flanking element and downstream by a second flanking element, wherein said first and second flanking element are different and wherein each of said first and second flanking element are homologous to a continuous DNA sequence on either side of the double-strand break introduced in (a) of  claim 8  within said target sequence in the genome of said eukaryotic cell.   
     
     
         10 . The method of  claim 8 , wherein said cell is analysed for successful modification of said target sequence in the genome. 
     
     
         11 . The method of  claim 8 , wherein the cell is selected from the group consisting of a mammalian or vertebrate cell, a plant cell or a fungal cell. 
     
     
         12 . The method of  claim 8 , wherein the cell is an oocyte. 
     
     
         13 . A method of producing a non-human vertebrate or mammal carrying a modified target sequence in its genome, the method comprising transferring a cell produced by the method of  claim 9  into a pseudo pregnant female host. 
     
     
         14 . The method of  claim 8 , wherein the cell is selected from the group consisting of rodents, dogs, felides, primates, rabbits, pigs, cows, chickens, turkeys, pheasants, ducks, geese, quails, ostriches, emus, cassowaries and zebrafish. 
     
     
         15 . A method of producing a protein or fusion protein having the activity of an endonuclease encoded by the nucleic acid molecule of  claim 1  comprising the steps of: (a) culturing a host cell comprising the nucleic acid molecule of  claim 1  and (b) isolating the produced protein or fusion protein. 
     
     
         16 . A host cell comprising the vector of  claim 5 . 
     
     
         17 . A protein or fusion protein having the activity of an endonuclease encoded by the vector of  claim 5 .