Modular DNA-binding domains and methods of use
The present invention refers to methods for selectively recognizing a base pair in a DNA sequence by a polypeptide, to modified polypeptides which specifically recognize one or more base pairs in a DNA sequence and, to DNA which is modified so that it can be specifically recognized by a polypeptide and to uses of the polypeptide and DNA in specific DNA targeting as well as to methods of modulating expression of target genes in a cell.
1. A method for producing a nucleic acid molecule encoding an engineered transcription activator-like (TAL) effector fusion protein that selectively binds a predetermined target DNA sequence in a mammalian cell, the method comprising:
(a) combining nucleic acid segments encoding portions of a TAL effector repeat domain to produce a nucleotide sequence encoding an engineered TAL effector repeat domain capable of selectively binding a predetermined target DNA sequence in a mammalian cell, wherein
i, a TAL effector repeat domain comprises repeat units, wherein the repeat units comprise hypervariable regions which collectively recognize a target DNA sequence and
ii. the nucleic acid segments are assembled in an order based on the sequence of base pairs present in the predetermined target DNA sequence and on the hypervariable regions; and
(b) ligating the nucleic acid segments to produce a nucleic acid molecule encoding a TAL effector fusion protein, wherein the TAL effector fusion protein comprises
i. the engineered TAL effector repeat domain and
ii. at least one additional domain
wherein the engineered TAL effector repeat domain is capable of selectively binding a predetermined target DNA sequence in a mammalian cell.
2. The method of claim 1 , wherein at least one of the hypervariable regions comprises a member selected from the group consisting of:
(a) HD for recognition of C/G;
(b) NI for recognition of A/T;
(c) NG for recognition of T/A;
(d) NS for recognition of C/G or A/T or T/A or G/C;
(e) NN for recognition of G/C or A/T;
(f) IG for recognition of T/A;
(g) N for recognition of C/G;
(h) HG for recognition of C/G or T/A; and
(i) H for recognition of T/A.
3. The method of claim 1 , wherein the hypervariable regions correspond to amino acids 12 and 13 in the repeat units.
4. The method of claim 1 , wherein the repeat domain comprises 1.5 to 40.5 repeat units.
5. The method of claim 1 , wherein the repeat domain comprises 11.5 to 33.5 repeat units.
6. The method of claim 1 , wherein the additional domain comprises a bacterial, viral, fungal, oomycete, human, animal, plant, or artificial protein, or part thereof.
7. The method of claim 1 , wherein the additional domain comprises a protein or functional part or domain thereof, that is capable of modifying DNA or RNA.
8. The method of claim 1 , wherein the additional domain comprises a protein or functional part or domain thereof selected from the group consisting of: a transcription activator, a transcription repressor, a resistance-mediating protein, a nuclease, a topoisomerase, a ligase, an integrase, a recombinase, a resolvase, a methylase, an acetylase, a demethylase, and a deacetylase.
9. The method of claim 1 , wherein the repeat unit comprises 30 to 40 amino acids.
10. The method of claim 1 , wherein the repeat unit comprises 33, 34, 35 or 39 amino acids.
11. The method of claim 1 , wherein the nucleic acid molecule is a vector.
12. The method of claim 1 , wherein the additional domain is a nuclease domain.
13. The method of claim 1 , wherein the additional domain is an endonuclease domain.
14. The method of claim 1 , wherein the additional domain comprises a fungal, animal, plant, or artificial protein, or part thereof.
15. The method of claim 1 , wherein the target DNA sequence is a naturally occurring DNA sequence in an animal.