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 - 17 . (canceled) 
     
     
         18 . A method for producing a genetically-modified eukaryotic cell including an exogenous sequence of interest inserted in a chromosome of said eukaryotic cell, comprising:
 transfecting a 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; and   wherein said meganuclease is a recombinant single-chain meganuclease of  claim 38 .   
     
     
         19 . A method as in  claim 18  wherein:
 said second nucleic acid further comprises sequences homologous to sequences flanking said cleavage site and said sequence of interest is inserted at said cleavage site by homologous recombination. 
 
     
     
         20 . A method as in  claim 18  wherein:
 said second nucleic acid lacks substantial homology to said cleavage site and said sequence of interest is inserted into said chromosome by non-homologous end joining. 
 
     
     
         21 . A method for producing a genetically-modified eukaryotic cell including an exogenous sequence of interest inserted in a chromosome of said eukaryotic cell, comprising:
 introducing a meganuclease protein into a eukaryotic cell; and   transfecting said eukaryotic cell with a nucleic acid 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; and   wherein said meganuclease is a recombinant single-chain meganuclease of  claim 38 .   
     
     
         22 . A method as in  claim 21  wherein:
 said nucleic acid further comprises sequences homologous to sequences flanking said cleavage site and said sequence of interest is inserted at said cleavage site by homologous recombination. 
 
     
     
         23 . A method as in  claim 21  wherein:
 said nucleic acid lacks substantial homology to said cleavage site and said sequence of interest is inserted into said chromosome by non-homologous end joining. 
 
     
     
         24 . A method for producing a genetically-modified eukaryotic cell by disrupting a target sequence in a chromosome of said eukaryotic cell, comprising:
 transfecting a eukaryotic cell with a nucleic acid encoding a meganuclease;   wherein said meganuclease produces a cleavage site in said chromosome and said target sequence is disrupted by non-homologous end joining at said cleavage site; and   wherein said meganuclease is a recombinant single-chain meganuclease of  claim 38 .   
     
     
         25 . A method of producing a genetically-modified non-human organism comprising:
 producing a genetically-modified non-human eukaryotic cell according to the method of  claim 24 ; and   growing said genetically-modified non-human eukaryotic cell to produce said genetically-modified non-human organism.   
     
     
         26 . A method as in  claim 25  wherein:
 said non-human eukaryotic cell is selected from the group consisting of a gamete, a zygote, a blastocyst cell, an embryonic stem cell, and a protoplast cell. 
 
     
     
         27 - 37 . (canceled) 
     
     
         38 . 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 first and second subunits are capable of functioning together to recognize and cleave a non-palindromic DNA sequence.   
     
     
         39 . The recombinant single-chain meganuclease of  claim 38 , wherein said polypeptide linker comprises at least 50% of polar uncharged amino acids. 
     
     
         40 . The recombinant single-chain meganuclease of  claim 41 , wherein said polypeptide linker comprises from the N-terminus to the C-terminus a first loop, a first α-helix, a first turn, a second α-helix, and a second loop. 
     
     
         41 . The recombinant single-chain meganuclease of  claim 38 , wherein said polypeptide linker comprises:
 (a) an SX 1 PGSX 2 GGX 3  (SEQ ID NO:111) motif, wherein X 1  is L or blank, X 2  is V or L, and X 3  is L, I, or V;   (b) an SPSQASSAASS (SEQ ID NO:112) motif, an SPSQASSAASSASSS (SEQ ID NO:113) motif, or an SPSQASSAASSASSAASS (SEQ ID NO:114) motif;   (c) a PGSGX 4  (SEQ ID NO:115) motif, wherein X 4  is I, T, P, V, L, S or A; and   (d) an SEAX 5 RAGX 6 A (SEQ ID NO:116) motif or an SEAX 5 RASSAGX 6 A (SEQ ID NO:117) motif, wherein X 5  is L, T, S, or I, and wherein X 6  is blank or G.   
     
     
         42 . The recombinant single-chain meganuclease of  claim 40 , wherein:
 (a) said first loop comprises said SX 1 PGSX 2 GGX 3  (SEQ ID NO:111) motif;   (b) said first α-helix comprises said SPSQASSAASS (SEQ ID NO:112) motif, said SPSQASSAASSASSS (SEQ ID NO:113) motif, or said SPSQASSAASSASSAASS (SEQ ID NO:114) motif;   (c) said first turn comprises said PGSGX 4  (SEQ ID NO:115) motif; and   (d) said SEAX 5 RAGX 6 A (SEQ ID NO:116) motif or said SEAX 5 RASSAGX 6 A (SEQ ID NO:117) motif spans the 3′ end of said second α-helix and the 5′ end of said second loop.   
     
     
         43 . The recombinant single-chain meganuclease of  claim 40 , wherein said polypeptide linker comprises a C-capping motif spanning the 3′ end of said second α-helix and the 5′ end of said second loop, wherein said C-capping motif comprises an AX 7 RAGX 8 A (SEQ ID NO:118) motif, wherein X 7  is L, T, S, or I, and wherein X 8  is blank or G.