Patent ID: 9340777
Filing Date: 2016-05-17
CPC Classification: A61P,C12N,C12Y

Claim Text:
1. A method for producing an isolated genetically-modified eukaryotic cell including an exogenous sequence of interest inserted in a chromosome of said isolated genetically-modified eukaryotic cell, comprising: transfecting an isolated eukaryotic cell with one or more nucleic acids including: (i) a first nucleic acid sequence encoding a meganuclease, and (ii) a second nucleic acid sequence including said sequence of interest; wherein said meganuclease produces a cleavage site in said chromosome and said sequence of interest is inserted into said chromosome at said cleavage site, thereby producing said isolated genetically-modified eukaryotic cell including said exogenous sequence of interest inserted in said chromosome of said isolated genetically-modified eukaryotic cell; and wherein said meganuclease is a recombinant single-chain meganuclease comprising: (a) a first subunit comprising a polypeptide having at least 85% sequence identity to residues 9-151 of SEQ ID NO: 1; (b) a second subunit comprising a polypeptide having at least 85% sequence identity to residues 9-151 of SEQ ID NO: 1; and (c) a polypeptide linker, said polypeptide linker being covalently bound to said first and said second subunit; said first subunit being covalently bound to said polypeptide linker at a residue corresponding to a position selected from the group of positions 153-163 of SEQ ID NO: 1; said second subunit being covalently bound to said polypeptide linker at a residue corresponding to a position selected from the group of positions 1-9 of SEQ ID NO: 1; wherein said polypeptide linker is SLPGSVGGLSPSQASSAASSASSSPGSGISEALRAGATKS (SEQ ID NO: 77); wherein said first and second subunits are capable of functioning together to recognize and cleave a non-palindromic DNA sequence.