Methods and compositions for alteration of a cystic fibrosis transmembrane conductance regulator (CFTR) gene

1. A protein comprising an engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four, five or six zinc finger recognition regions ordered F1 to F4, F1 to F5 or F1 to F6 from N-terminus to C-terminus, and wherein the protein is selected from the group consisting of:

(i) a 5-finger zinc finger protein comprising the following recognition regions:

F1: WPSCLYA (SEQ ID NO:8);

F2: NGVLLKR (SEQ ID NO:9);

F3: QSGNLAR (SEQ ID NO:10);

F4: RSDNLSE (SEQ ID NO:11); and

F5: NPRNRFT (SEQ ID NO:12), wherein the protein binds to a target site as shown in SEQ ID NO:37;

(ii) a 5-finger zinc finger protein comprising the following recognition regions:

F1: RSDVLSE (SEQ ID NO:13);

F2: QSGNLAR (SEQ ID NO:10);

F3: QSGHLSR (SEQ ID NO:14);

F4: RSDVLSE (SEQ ID NO:15); and

F5: WSASLSK (SEQ ID NO:16), wherein the protein binds to a target site as shown in SEQ ID NO:38;

(iii) a 4-finger zinc finger protein comprising the following recognition regions:

F1: QNATRIN (SEQ ID NO:17);

F2: QSGNLAR (SEQ ID NO:10);

F3: RSDNLST (SEQ ID NO:18); and

F4: QSADRKK (SEQ ID NO:19), wherein the protein binds to a target site as shown in SEQ ID NO:39;

(iv) a 6-finger zinc finger protein comprising the following recognition regions:

F1: TNQNRIT (SEQ ID NO:20);

F2: RNQTRIT (SEQ ID NO:21);

F3: QSGNLAR (SEQ ID NO:10);

F4: QSNTRIM (SEQ ID NO:22);

F5: TSGNLTR (SEQ ID NO:23); and

F6: QSNALHQ (SEQ ID NO:24), wherein the protein binds to a target site as shown in SEQ ID NO:40;

(v) a 5-finger zinc finger protein comprising the following recognition regions:

F1: TSSDRKK (SEQ ID NO:25);

F2: QSSDLSR (SEQ ID NO:26);

F3: DRSNLTR (SEQ ID NO:27);

F4: TSGNLTR (SEQ ID NO:23); and

F5: WRLSLQV (SEQ ID NO:28), wherein the protein binds to a target site as shown in SEQ ID NO:41;

(vi) a 6-finger zinc finger protein comprising the following recognition regions:

F1: QSGNLAR (SEQ ID NO:10);

F2: QGANLIK (SEQ ID NO:29);

F3: RSDHLSA (SEQ ID NO:30);

F4: ESRYLMV (SEQ ID NO:31);

F5: RSDNLST (SEQ ID NO:18); and

F6: DRSNRKT (SEQ ID NO:32), wherein the protein binds to a target site as shown in SEQ ID NO:42;

(vii) a 5-finger zinc finger protein comprising the following recognition regions:

F1: TSGNLTR (SEQ ID NO:23);

F2: QSNALHQ (SEQ ID NO:24);

F3: QSGNLAR (SEQ ID NO:10);

F4: TSGNLTR (SEQ ID NO:23); and

F5: WWTSRAL (SEQ ID NO:33), wherein the protein binds to a target site as shown in SEQ ID NO:43; and

(viii) a 4-finger zinc finger protein comprising the following recognition regions:

F1: HSNARKT (SEQ ID NO:34);

F2: TSGNLTR (SEQ ID NO:23);

F3: TLQNRMS (SEQ ID NO:35); and

F4: DQSTLRN (SEQ ID NO:36), wherein the protein binds to a target site as shown in SEQ ID NO:44.

2. A fusion protein comprising a protein according to claim 1 and a cleavage domain.

3. The fusion protein of claim 2 , wherein the cleavage domain is a cleavage half-domain.

4. The fusion protein of claim 3 , wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.

5. The fusion protein of claim 3 , wherein the cleavage half-domain is an engineered FokI cleavage half-domain.

6. A polynucleotide encoding the protein of claim 1 .

7. An isolated cell comprising the protein of claim 1 .

8. An isolated cell comprising the polynucleotide of claim 6 .

9. The cell of claim 7 , wherein the cell is selected from the group consisting of an embryonic stem cell (ESC), a hematopoietic stem cell, a nerve stem cell, a skin stem cell, a muscle stem cell, a lung stem cell, an induced pluripotent stem cell (iPSC) and a fibroblast cell.

10. The cell of claim 8 , wherein the cell is selected from the group consisting of an embryonic stem cell (ESC), a hematopoietic stem cell, a nerve stem cell, a skin stem cell, a muscle stem cell, a lung stem cell, an induced pluripotent stem cell (iPSC) and a fibroblast cell.

11. A method of modifying a cystic fibrosis transmembrane conductance regulator (CFTR) gene in an isolated cell, the method comprising;

cleaving the CFTR gene with one or more fusion proteins according to claim 2 .

12. The method of claim 11 , wherein the modification is selected from the group consisting of an insertion, a deletion, a substitution and combinations thereof.

13. The method of claim 11 , further comprising introducing an exogenous sequence into the CFTR gene.

14. The method of claim 11 , wherein the modification corrects a mutation in the CFTR gene.

15. The method of claim 14 , wherein the mutation is selected from the group consisting of ΔF508, ΔI507 and combinations thereof.

16. A method of generating a model system for the study of cystic fibrosis (CF), the method comprising modifying cells according to the method of claim 11 .

17. The method of claim 16 , wherein the model system comprises a cell line.

18. The method of claim 16 , wherein the model system comprises a non-human animal.

19. A kit comprising the protein according to claim 1 .