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 producing a dimer, comprising expressing in a living cell:
(a) a nucleic acid encoding a first transcription activator-like (TAL) effector endonuclease monomer comprising a plurality of TAL effector repeat sequences and a FokI endonuclease domain, wherein each of said plurality of TAL effector repeat sequences comprises a repeat-variable diresidue, and wherein said first TAL effector endonuclease monomer is capable of binding to a first half-site sequence of a target DNA, and
(b) a nucleic acid encoding a second TAL effector endonuclease monomer comprising a plurality of TAL effector repeat sequences and a FokI endonuclease domain, wherein each of said plurality of TAL effector repeat sequences of said second TAL effector endonuclease monomer comprises a repeat-variable diresidue, and wherein said second TAL effector endonuclease monomer 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, wherein said first TAL effector endonuclease monomer and said second endonuclease monomer form a dimer, and wherein said dimer is capable of cleaving said target DNA within 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. 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 in a vector.
3. 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 in a plasmid.
4. The method of claim 1 , wherein said spacer sequence is 12 to 30 nucleotides in length.
5. The method of claim 1 , wherein said spacer sequence is 18 nucleotides in length.
6. The method of claim 1 , wherein said first and second TAL effector endonuclease monomers each comprise 15 or more TAL effector repeat sequences.
7. 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.