Tal effector-mediated dna modification

Abstract

Materials and Methods related to gene targeting (e.g., gene targeting with transcription activator-like effector nucleases; “TALENS”) are provided.

1 . (canceled) 
     
     
         2 . A first transcription activator-like (TAL) effector endonuclease monomer comprising:
 (i) a FokI endonuclease domain, and   (ii) a TAL effector domain comprising a plurality of TAL effector repeat sequences, wherein the plurality of TAL effector repeat sequences of the first TAL effector endonuclease monomer, in combination, bind to a first nucleotide sequence in a target DNA sequence in a cell,   wherein the first TAL effector endonuclease monomer is capable of forming a dimer with a second TAL effector endonuclease monomer comprising a FokI endonuclease domain and a TAL effector domain having a plurality of TAL effector repeat sequences that, in combination, bind to a second nucleotide sequence in the target DNA sequence, wherein the dimer is formed between the FokI domain of the first TAL effector endonuclease monomer and the FokI domain of the second TAL effector endonuclease monomer when the TAL effector domain of the first TAL effector endonuclease monomer is bound to the first nucleotide sequence and the TAL effector domain of the second TAL effector endonuclease monomer is bound to the second nucleotide sequence,   wherein the first nucleotide sequence and the second nucleotide sequence are different and are separated by a spacer sequence, and   wherein the dimer cleaves the target DNA sequence within the cell.   
     
     
         3 . The first TAL effector endonuclease monomer of  claim 2 , wherein the target DNA sequence is in a promoter region. 
     
     
         4 . The first TAL effector endonuclease monomer of  claim 2 , wherein the TAL effector domain comprises 15 or more DNA binding repeats. 
     
     
         5 . The first TAL effector endonuclease monomer of  claim 2 , wherein each DNA binding repeat comprises a repeat variable-diresidue (RVD) that determines recognition of a base pair in the target DNA sequence, wherein each DNA binding repeat is responsible for recognizing one base pair in the target DNA sequence, and wherein the RVD comprises one or more of:
 HD for recognizing C;   NG for recognizing T;   NI for recognizing A;   NN for recognizing G;   NS for recognizing A;   HG for recognizing T;   IG for recognizing T;   NK for recognizing G;   HA for recognizing C;   ND for recognizing C;   HI for recognizing C;   HN for recognizing G; and   NA for recognizing G.   
     
     
         6 . The first TAL effector endonuclease monomer of  claim 5 , wherein each DNA binding repeat comprises a RVD that determines recognition of a base pair in the target DNA sequence, wherein each DNA binding repeat is responsible for recognizing one base pair in the target DNA sequence, and wherein the RVD comprises one or more of:
 HA for recognizing C;   ND for recognizing C;   HI for recognizing C;   HN for recognizing G;   NA for recognizing G; and   NK for recognizing G;   
       and one or more of:
 HD for recognizing C; 
 NG for recognizing T; 
 NI for recognizing A; 
 NN for recognizing G or A; 
 NS for recognizing A or C or G or T; 
 HG for recognizing T; and 
 IG for recognizing T. 
 
     
     
         7 . The first TAL effector endonuclease monomer of  claim 1 , wherein the spacer sequence is 12 to 30 nucleotides in length. 
     
     
         8 . The first TAL effector endonuclease monomer of  claim 1 , wherein the spacer sequence is 18 nucleotides in length.