Methods and compositions for inactivating alpha 1,6 fucosyltransferase (
Disclosed herein are methods and compositions for inactivating a FUT8 gene, using fusion proteins comprising a zinc finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding said fusion proteins are also provided, as are cells comprising said polynucleotides and fusion proteins.
1. A zinc finger DNA-binding domain, wherein the DNA-binding domain comprises six zinc finger recognition regions ordered F1 to F6 from N-terminus to C-terminus, and wherein F1, F2, F4, F5 and F6 comprise the following amino acid sequences:
F1: RSDNLSV (SEQ ID NO:19);
F2: QNATRIN (SEQ ID NO:15);
F4: QSATRTK (SEQ ID NO:21);
F5 RSDNLSR (SEQ ID NO:22); and
F6: RNDNRKT (SEQ ID NO:23).
2. The zinc finger DNA-binding domain of claim 1 , wherein F3 comprises RSDNLST (SEQ ID NO:20).
3. The zinc finger DNA-binding domain of claim 1 , wherein F3 comprises RSDHLSQ (SEQ ID NO:24).
4. A fusion protein comprising a zinc finger DNA-binding domain according to claim 1 and at least one cleavage domain or at least one cleavage half-domain.
5. The fusion protein of claim 4 , wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.
6. The fusion protein of claim 5 , wherein the cleavage half-domain is an engineered FokI cleavage half-domain.
7. An isolated polynucleotide encoding the zinc finger DNA-binding domain of claim 1 .
8. A polynucleotide encoding the protein according to claim 4 .
9. An isolated cell comprising a protein according to claim 4 .
10. The cell of claim 9 , wherein Fut8 is partially inactivated.
11. The cell of claim 9 , wherein Fut8 is fully inactivated.
12. The cell of claim 9 , wherein one or more additional genes are partially or fully inactivated.
13. The cell of claim 12 , wherein dihydrofolate reductase (DHFR) and glutamine synthetase (GS) have been inactivated.
14. A zinc finger DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition regions ordered F1 to F4 from N-terminus to C-terminus, and wherein F1, F2, F3, and F4 comprise the following amino acid sequences:
F1: RSDVLSA (SEQ ID NO:14);
F2: QNATRIN (SEQ ID NO:15);
F3: DRSNLSR (SEQ ID NO:16); and
F4: RLDNRTA (SEQ ID NO:17).
15. A fusion protein comprising a zinc finger DNA-binding domain according to claim 14 and at least one cleavage domain or at least one cleavage half-domain.
16. The fusion protein of claim 15 , wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.
17. The fusion protein of claim 16 , wherein the cleavage half-domain is an engineered FokI cleavage half-domain.
18. An isolated polynucleotide encoding the protein according to claim 14 .
19. A polynucleotide encoding the protein according to claim 15 .
20. An isolated cell comprising a protein according to claim 15 .
21. The cell of claim 20 , wherein Fut8 is partially inactivated.
22. The cell of claim 20 , wherein Fut8 is fully inactivated.
23. The cell of claim 20 , wherein one or more additional genes are partially or fully inactivated.
24. The cell of claim 23 , wherein dihydrofolate reductase (DHFR) and glutamine synthetase (GS) have been inactivated.