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 I-CreI meganuclease variant, comprising the following substitutions: K28Q, Q38K and S40R of the wild-type I-CreI (SEQ ID NO: 1), said variant being able to cleave a DNA target sequence consisting of a mutant I-CreI site wherein at least the nucleotide A in position -7 and/or -6, and/or the nucleotide T in position +6 and/or +7 has been replaced with a different nucleotide. 
     
     
         2 . The I-CreI meganuclease variant according to  claim 1 , said variant having an arginine (R) or a lysine (K) in position 38, and being able to cleave a DNA target sequence consisting of a mutant I-CreI site comprising a guanine in position -7 and/or a cytosine in position +7. 
     
     
         3 . The I-CreI variant according to  claim 1 , comprising one or more additional mutation(s) at positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target. 
     
     
         4 . The I-CreI variant according to  claim 3 , wherein, said mutations are in positions selected from the group consisting of: 124, Q26, S32, Q44, R68, R70, D75, 177 and T140. 
     
     
         5 . The I-CreI variant according to  claim 4 , comprising the replacement of the aspartic acid in position 75 with an uncharged amino acid. 
     
     
         6 . The I-CreI variant according to  claim 5 , comprising the D75N or the D75V mutation. 
     
     
         7 . The I-CreI variant according to  claim 4 , comprising the R70S mutation. 
     
     
         8 . The I-CreI variant according to  claim 1 , comprising one or more additional mutation(s) in positions 80 to 163 of I-CreI. 
     
     
         9 . The I-CreI variant of  claim 1 , which is a homodimer. 
     
     
         10 . The I-CreI variant of  claim 1 , which is a heterodimer comprising monomers from two different variants. 
     
     
         11 . A single-chain chimeric endonuclease comprising the fusion of a monomer from a variant as defined in  claim 1 , with a monomer or a domain from a LAGLIDADG homing endonuclease. 
     
     
         12 . A I-CreI meganuclease variant having a modified cleavage specificity, wherein
 (a) at least one of the amino acids K28 and/or S40 from the 13 1 132 hairpin of the wild-type I-CreI (SEQ ID NO: 1) has been substituted with an amino acid selected from the group consisting of A, C, D, E, G, H, K, N, P, Q, R, S, T, L, V, W and Y and optionally   (b) at least one of the amino acids N30, Y33 and Q38 from the 13 1 132 hairpin of the wild-type I-CreI (SEQ ID NO: 1) has been substituted with an amino acid selected from the group consisting of A, C, D, E, G, H, K, N, P, Q, R, S, T, L, V, W and Y; and   wherein the I-CreI variant is able to cleave a DNA target sequence consisting of a mutant I-CreI site wherein at least the nucleotide doublet AA in positions -7 and -6 and/or the nucleotide doublet TT in positions +6 to +7 has been replaced with a different nucleotide doublet.   
     
     
         13 . The I-CreI homing endonuclease variant of  claim 1  wherein the amino acid S40 has been substituted.