TAL effector-mediated DNA modification
Materials and methods related to gene targeting (e.g., gene targeting with transcription activator-like effector nucleases; “TALENS”) are provided.
1. A method for making an article of manufacture comprising a nucleic acid encoding a first transcription activator-like (TAL) effector endonuclease monomer and a nucleic acid encoding a second TAL effector endonuclease monomer, wherein said method comprises:
(a) ligating a nucleotide sequence encoding a first TAL effector repeat domain comprising a plurality of TAL effector repeat sequences to a nucleotide sequence encoding a FokI endonuclease domain to form said nucleic acid encoding said first TAL effector endonuclease monomer, wherein said first TAL effector repeat domain is capable of binding to a first half-site sequence of a target DNA, and
(b) ligating a nucleotide sequence encoding a second TAL effector repeat domain comprising a plurality of TAL effector repeat sequences to a nucleotide sequence encoding a FokI endonuclease domain to form said nucleic acid encoding said second TAL effector endonuclease monomer, wherein said second TAL effector repeat domain is capable of binding to a second half-site sequence of said target DNA,
wherein said first half-site sequence and said second half-site sequence are different and are separated by a spacer sequence, and wherein said first TAL effector endonuclease monomer and said second TAL effector endonuclease monomer are capable of forming a dimer that cleaves said target DNA in a living cell when said first TAL effector endonuclease monomer is bound to said first half-site sequence and said second TAL effector endonuclease monomer is bound to said second half-site sequence.
2. A method of claim 1 , wherein said nucleic acid encoding said first TAL effector endonuclease monomer or said nucleic acid encoding said second TAL effector endonuclease monomer is in a vector.
3. A method of claim 1 , wherein said nucleic acid encoding said first TAL effector endonuclease monomer or said nucleic acid encoding said second TAL effector endonuclease monomer is in a plasmid.
4. The method of claim 1 , wherein said first and second TAL effector endonuclease monomers each comprise 15 or more TAL effector repeat sequences.
5. The method of claim 1 , wherein said nucleic acid encoding said first TAL effector endonuclease monomer or said nucleic acid encoding said second TAL effector endonuclease monomer is a mRNA.
6. The method of claim 1 , wherein said spacer sequence is 18 nucleotides in length.