Rationally-Designed Single-Chain Meganucleases With Non-Palindromic Recognition Sequences

Abstract

Disclosed are rationally-designed, non-naturally-occurring meganucleases in which a pair of enzyme subunits having specificity for different recognition sequence half-sites are joined into a single polypeptide to form a functional heterodimer with a non-palindromic recognition sequence. The invention also relates to methods of producing such meganucleases, and methods of producing recombinant nucleic acids and organisms using such meganucleases.

1 . 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 comprising 28-43 amino acids, 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 first and second subunits are capable of functioning together to recognize and cleave a non-palindromic DNA sequence.   
     
     
         2 . The recombinant single-chain meganuclease of claim  38 , wherein said polypeptide linker comprises at least 50% of polar uncharged amino acids.