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US 9,434,931 B2Granted
Rationally-designed single-chain meganucleases with non-palindromic recognition sequences
AssigneePrecision BioSciences, Inc.
InventorsJames Jefferson Smith, Derek Jantz
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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.
Claims4
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 28-45 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 polypeptide linker comprises an ALRAGA (SEQ ID NO:75) sequence;
- and
- wherein said first and second subunits are capable of functioning together to recognize and cleave a non-palindromic DNA sequence.
10 more elements
Dependencies are inferred from the claim text.