Engineered cleavage half-domains

Abstract

Disclosed herein are engineered cleavage half-domains; fusion polypeptides comprising these engineered cleavage half-domains; polynucleotides encoding the engineered cleavage half-domains and fusion proteins; and cells comprising said polynucleotides and/or fusion proteins. Also described are methods of using these polypeptides and polynucleotides, for example for targeted cleavage of a genomic sequence.

1 . A polypeptide comprising an engineered FokI cleavage half-domain, wherein
 (i) the engineered cleavage half-domain comprises a mutation in one or more wild-type amino acid residue 483, 486, 487, 490, 499, 538 or combinations thereof, wherein the amino acid residues are numbered relative to full length wild-type FokI;   (ii) the engineered cleavage half-domain forms an obligate heterodimer with a wild-type cleavage half-domain or a second engineered cleavage half-domain; and   (iii) if the engineered cleavage half-domain comprises a mutation at amino acid residue 490, it further comprises at least one additional amino acid mutation.   
     
     
         2 . The polypeptide of  claim 1 , wherein the amino acid residues at positions 490 and 538 are mutated. 
     
     
         3 . The polypeptide of the  claim 2 , wherein the engineered cleavage half-domain comprises the polypeptide designated E490K:I538K. 
     
     
         4 . The polypeptide of  claim 1 , wherein the wild-type amino acid at position 486 is mutated. 
     
     
         5 . The polypeptide of  claim 4 , wherein the engineered cleavage half-domain comprises the polypeptide designated Q486E. 
     
     
         6 . The polypeptide of  claim 1 , wherein the wild-type amino acid residue at position 499 is mutated. 
     
     
         7 . The polypeptide of  claim 6 , wherein the engineered cleavage half-domain comprises the polypeptide designated I499L. 
     
     
         8 . The polypeptide of  claim 7 , wherein the engineered cleavage half-domain comprises the polypeptide designated Q486E:I499L. 
     
     
         9 . A heterodimer comprising an engineered cleavage half-domain of  claim 1  and a wild-type FokI cleavage half-domain. 
     
     
         10 . The heterodimer of  claim 9 , wherein the engineered cleavage half-domain comprises the polypeptide designated E490K:I538K. 
     
     
         11 . A heterodimer comprising a first engineered cleavage half-domain of  claim 1  and a second engineered cleavage half-domain. 
     
     
         12 . The heterodimer of  claim 11 , wherein the first engineered cleavage half-domain comprises mutation in amino acid residue 490 and/or amino acid residue 538. 
     
     
         13 . The heterodimer of  claim 11 , wherein the second engineered cleavage half-domain comprises a mutation in amino acid residue 486 and/or amino acid residue residue 499. 
     
     
         14 . The polypeptide of  claim 1 , further comprising a zinc finger protein DNA-binding domain. 
     
     
         15 . A polynucleotide encoding the polypeptide of  claim 1 . 
     
     
         16 . A polynucleotide encoding the polypeptide of  claim 14 . 
     
     
         17 . An isolated cell comprising the polypeptide of  claim 1 . 
     
     
         18 . An isolated cell comprising the polynucleotide of  claim 15 . 
     
     
         19 . An isolated cell comprising the polynucleotide of  claim 16 . 
     
     
         20 . A method for cleaving genomic cellular chromatin in a region of interest, the method comprising:
 (a) selecting a first nucleotide sequence in the region if interest;   (b) engineering a first zinc finger binding domain to bind to the first sequence;   (c) expressing a first fusion protein in a cell, the first fusion protein comprising the engineered zinc finger binding domain and an engineered cleavage half-domain of  claim 1 ;   (d) expressing a second fusion protein in the cell, the second fusion protein comprising a second zinc finger binding domain and a second cleavage half-domain;   wherein the first fusion protein binds to the first nucleotide sequence, the second fusion protein binds to a second nucleotide sequence located between 2 and 50 nucleotides from the first nucleotide sequence, the first and second engineered cleavage domains form a heterodimer that cleaves the cellular chromatin in the region of interest.   
     
     
         21 . The method of  claim 20 , wherein the second cleavage half-domain is an engineered cleavage half-domain. 
     
     
         22 . The method of  claim 20 , wherein cleavage occurs between the first and second nucleotide sequences. 
     
     
         23 . The method of  claim 20 , wherein the second zinc finger binding domain is engineered to bind to the second nucleotide sequence. 
     
     
         24 . The method of  claim 20 , wherein the cellular chromatin is in a chromosome. 
     
     
         25 . The method of  claim 20 , further comprising contacting the cell with a polynucleotide comprising a third nucleotide sequence, wherein the third nucleotide sequence is homologous but non-identical with the first nucleotide sequence; wherein cleavage of the cellular chromatin in the region of interest facilitates homologous recombination between the first nucleotide sequence and the third nucleotide sequence, resulting in alteration of the first nucleotide sequence. 
     
     
         26 . The method of  claim 25 , wherein the third nucleotide sequence contains sequences not present in the region of interest that are flanked by sequences homologous to the region of interest. 
     
     
         27 . The method of  claim 20 , wherein the cell is arrested in the G2 phase of the cell cycle.