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; or   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.   
     
     
         3 . 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.   
     
     
         4 . 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 . 
 
     
     
         5 . 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 .   
     
     
         6 . 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 .   
     
     
         7 . A method for treating a disease by gene therapy in a eukaryote, comprising:
 transfecting at least one cell of said eukaryote with one or more nucleic acids including
 (i) a first nucleic acid sequence encoding a meganuclease, and 
 (ii) a second nucleic acid sequence including a 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;   wherein said meganuclease is a recombinant meganuclease of  claim 1 ; and   wherein insertion of said sequence of interest provides said gene therapy for said disease.   
     
     
         8 . A method for treating a disease by gene therapy in a eukaryote, comprising:
 introducing a meganuclease protein into at least one cell of said eukaryote; and   transfecting said eukaryotic cell with a nucleic acid including a 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;   wherein said meganuclease is a recombinant meganuclease of  claim 1 ; and   wherein insertion of said sequence of interest provides said gene therapy for said disease.   
     
     
         9 . A method for treating a disease by gene therapy in a eukaryote by disrupting a target sequence in a chromosome of said eukaryotic cell, comprising:
 transfecting at least one cell of said eukaryote 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;   wherein said meganuclease is a recombinant meganuclease of  claim 1 ; and   wherein disruption of said target sequence provides said gene therapy for said disease.