Rationally Designed Meganucleases With Altered Sequence Specificity and DNA-Binding Affinity

Abstract

Rationally-designed LAGLIDADG meganucleases and methods of making such meganucleases are provided. In addition, methods are provided for using the meganucleases to generate recombinant cells and organisms having a desired DNA sequence inserted into a limited number of loci within the genome, as well as methods of gene therapy, for treatment of pathogenic infections, and for in vitro applications in diagnostics and research.

1 . A recombinant meganuclease having altered specificity for at least one recognition sequence half-site relative to a wild-type I-CreI meganuclease, comprising:
 a polypeptide having at least 85% sequence similarity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO: 1; and   having specificity for a recognition sequence half-site which differs by at least one base pair from a half-site within an I-CreI meganuclease recognition sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5;   wherein said recombinant meganuclease comprises at least one modification of Table 1 which is not an excluded modification.   
     
     
         2 . A recombinant meganuclease having altered binding affinity for double-stranded DNA relative to a wild-type I-CreI meganuclease, comprising:
 a polypeptide having at least 85% sequence similarity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO: 1;   wherein DNA-binding affinity has been increased by at least one modification corresponding to a substitution selected from the group consisting of:   (a) substitution of E80, D137, I81, L112, P29, V64 or Y66 with H, N, Q, S, T, K or R; or   (b) substitution of T46, T140 or T143 with K or R.   
     
     
         3 . A recombinant meganuclease having altered binding affinity for double-stranded DNA relative to a wild-type I-CreI meganuclease, comprising:
 a polypeptide having at least 85% sequence similarity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO: 1;   wherein DNA-binding affinity has been decreased by at least one modification corresponding to a substitution selected from the group consisting of:   (a) substitution of K34, K48, R51, K82, K116 or K139 with H, N, Q, S, T, D or E; or   (b) substitution of I81, L112, P29, V64, Y66, T46, T140 or T143 with D or E.   
     
     
         4 . A recombinant meganuclease monomer having altered affinity for dimer formation with a reference meganuclease monomer, comprising:
 a polypeptide having at least 85% sequence similarity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO: 1;   wherein affinity for dimer formation has been altered by at least one modification corresponding to a substitution selected from the group consisting of:   (a) substitution of K7, K57 or K96 with D or E; or   (b) substitution of E8 or E61 with K or R.   
     
     
         5 . A recombinant meganuclease heterodimer comprising:
 a first polypeptide having at least 85% sequence similarity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO: 1;   wherein affinity for dimer formation has been altered by at least one modification corresponding to a substitution selected from the group consisting of:   (a) substitution of K7, K57 or K96 with D or E; and   a second polypeptide having at least 85% sequence similarity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO: 1;   wherein affinity for dimer formation has been altered by at least one modification corresponding to a substitution selected from the group consisting of:   (b) substitution of E8 or E61 with K or R.   
     
     
         6 . A recombinant meganuclease monomer or heterodimer of  claim 5 , further comprising:
 at least one modification selected from Table 1;   wherein said modification is not an excluded modification.   
     
     
         7 . 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 meganuclease of  claim 1 . 
   
     
     
         8 . A method as in  claim 7  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. 
 
     
     
         9 . A method as in  claim 7  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 
 
     
     
         10 . 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 meganuclease of  claim 1 .   
     
     
         11 . A method as in  claim 10  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. 
 
     
     
         12 . A method as in  claim 10  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 
 
     
     
         13 . 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 meganuclease of  claim 1 .   
     
     
         14 . A method of producing a genetically-modified organism comprising:
 producing a genetically-modified eukaryotic cell according to the method of  claim 7 ; and   growing said genetically-modified eukaryotic cell to produce said genetically-modified organism.   
     
     
         15 . A method as in  claim 14  wherein:
 said eukaryotic cell is selected from the group consisting of a gamete, a zygote, a blastocyst cell, an embryonic stem cell, and a protoplast cell.