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 modifying the genetic material of a cell, comprising:
(a) providing a cell containing a target DNA sequence; and
(b) introducing a first transcription activator-like (TAL) effector endonuclease monomer and a second TAL effector endonuclease monomer into the cell, each 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 specific nucleotide sequence in the target DNA sequence, wherein the plurality of TAL effector repeat sequences of the second TAL effector endonuclease monomer, in combination, bind to a second specific nucleotide sequence in the target DNA sequence, and wherein the first specific nucleotide sequence and the second specific nucleotide sequence are different and are separated by a spacer sequence,
such that the FokI endonuclease domain of the first TAL effector endonuclease monomer and the FokI endonuclease domain of the second TAL effector endonuclease monomer form a dimer that cleaves the target DNA sequence within the cell or progeny thereof when the TAL effector domain of the first TAL effector endonuclease monomer is bound to the first specific nucleotide sequence and the TAL effector domain of the second TAL effector endonuclease monomer is bound to the second specific nucleotide sequence.
2. The method of claim 1 , further comprising providing to the cell a nucleic acid comprising a sequence homologous to at least a portion of the target DNA sequence, such that homologous recombination occurs between the target DNA sequence and the nucleic acid.
3. The method of claim 1 , wherein the cell is a eukaryotic cell.
4. The method of claim 1 , wherein the cell is a mammalian cell.
5. The method of claim 1 , wherein the cell is a plant cell.
6. The method of claim 1 , wherein the cell is a prokaryotic cell.
7. The method of claim 1 , wherein the target DNA is chromosomal DNA.
8. The method of claim 1 , wherein the introducing comprises transfecting the cell with a nucleic acid encoding the first or second TAL effector-endonuclease monomer.
9. The method of claim 1 , wherein the introducing comprises mechanically injecting the first or second TAL effector-endonuclease monomer into the cell as a protein.
10. The method of claim 1 , wherein the introducing comprises delivering the first or second TAL effector-endonuclease monomer into the cell as a protein by means of the bacterial type III secretion system.
11. The method of claim 1 , wherein the introducing comprises introducing the first or second TAL effector-endonuclease monomer into the cell as a protein by electroporation.
12. The method of claim 1 , wherein the TAL effector domain that binds to a specific nucleotide sequence within the target DNA comprises 15 or more DNA binding repeats.
13. The method of claim 12 , 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.
14. The method of claim 13 , 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.
15. The method of claim 1 , further comprising generating a genetically modified organism comprising the cell or progeny thereof.
16. The method of claim 15 , wherein the organism is a plant.
17. The method of claim 16 , wherein the method further comprises obtaining seeds from the plant.
18. The method of claim 15 , wherein the organism is an animal.
19. The method of claim 1 , wherein the spacer sequence is 12 to 30 nucleotides in length.
20. The method of claim 1 , wherein the spacer sequence is 18 nucleotides in length.
21. The method of claim 5 , wherein the plant cell is a protoplast.
22. The method of claim 8 , wherein the nucleic acid is a vector.
23. The method of claim 8 , wherein the nucleic acid is a mRNA.