Obligate heterodimer variants of foki cleavage domain

Abstract

Disclosed are methods of making and using engineered Fold cleavage domain variants. Also disclosed are methods, compositions and fusion proteins containing obligate heterodimers of engineered Fold cleavage domain variants and DNA binding domains, such as zinc finger protein (ZFP) domains and transcription activator-like effector (TALE) domains.

1 - 27 . (canceled) 
     
     
         28 . A method for delivering a targeted genomic double-strand break (DSB) in cells, including plant, animal and human cells, comprising: (a) selecting a first nucleotide sequence in the region of 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 FokI cleavage domain variant 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 FokI cleavage domain variant; wherein the first fusion protein binds to the first nucleotide sequence, the second fusion protein binds to a second nucleotide sequence from the first nucleotide sequence on the complementary strand of DNA, the first and second engineered cleavage domains form a heterodimer that cleaves the cellular chromatin in the region of interest. 
     
     
         29 . The method of  claim 28 , wherein the human cells comprise sensitive human primary cells, human embryonic stem cells (hESC), adult human stem cells, human stem progenitor cells (hSPC) and human induced pluripotent stem cells (hiPSC). 
     
     
         30 . The method of  claim 28 , further comprising delivering either stimulate local homologous recombination (HR) with investigator-provided donor DNA or inducing gene mutations at the site of cleavage in the absence of a donor by non-homologous end joining (NHEJ) in cells. 
     
     
         31 . A method of cleaving a gene of interest in a cell, the method comprising: providing a fusion protein comprising a DNA binding domain and an engineered FokI cleavage domain variant of claim  1 , wherein the DNA binding domain binds to a target site in the gene of interest; and contacting the cell with the fusion protein under conditions such that the gene of interest is cleaved. 
     
     
         32 . The method of  claim 31 , wherein the gene of interest is CCR5, the DNA binding domain binds to a target site in the CCR5 gene, and the CCR5 gene is cleaved. 
     
     
         33 . The method of  claim 31 , wherein the DNA binding domain comprises zinc finger protein (ZFP) domain or transcription activator-like effector (TALE) domain. 
     
     
         34 . The method of  claim 31 , wherein the fusion protein includes 3- or 4-zinc finger proteins (ZFPs) that target CCR5 of human cells, and the obligate heterodimer comprises a first monomer containing the polypeptide designated D483R:Q486E:I499L, and a second monomer containing the polypeptide designated R487D:E490K:I538K. 
     
     
         35 . The method of  claim 34 , wherein the zinc finger proteins (ZFPs) may include ZF1, ZF2, ZF3, ZF4, ZF5 or ZF6, described in Table 1. 
     
     
         36 . A composition for disrupting a gene of interest in a cell, comprising an engineered fusion protein comprising a DNA binding domain to bind a gene of interest and an engineered FokI cleavage domain variant of claim  1 , wherein the fusion protein binds to and cleaves the gene of interest. 
     
     
         37 . The composition of  claim 36 , wherein the gene of interest is CCR5, the DNA binding domain binds to a target site in the CCR5 gene, and the CCR5 gene is cleaved. 
     
     
         38 . The composition of  claim 36 , wherein the DNA binding domain comprises zinc finger protein (ZFP) domain or transcription activator-like effector (TALE) domain. 
     
     
         39 . The composition of  claim 36 , wherein the fusion protein includes 3- or 4-zinc finger proteins (ZFPs) that target CCR5 of human cells, and the obligate heterodimer comprises a first monomer containing the polypeptide designated D483R:Q486E:I499L, and a second monomer containing the polypeptide designated R487D:E490K:I538K. 
     
     
         40 . The composition of  claim 39 , wherein the zinc finger proteins (ZFPs) may include ZF1, ZF2, ZF3, ZF4, ZF5 or ZF6, described in Table 1.