Methods and compositions for modification of the
Nucleases and methods of using these nucleases for modification of an HPRT locus and for increasing the frequency of gene modification at a targeted locus and clones and for generating animals.
1. A non-naturally occurring fusion protein comprising a transcription activator like effector domain DNA binding protein (TALE) that binds to a target site in an endogenous hypoxanthine guanine phosphoribosyltransferase (HPRT) gene comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs:69-78 and a nuclease cleavage domain, wherein the TALE domain comprises a repeat variable diresidue (RVD) sequence in the order presented and selected from a single row of Table 4.
2. A polynucleotide encoding the non-naturally occurring fusion proteins of claim 1 .
3. An isolated cell comprising the fusion proteins of claim 1 .
4. An isolated cell comprising the polynucleotides of claim 2 .
5. The isolated cell of claim 3 , wherein the cell is selected from the group consisting of a T-cell, a B-cell or a stem cell.
6. The isolated cell of claim 5 , wherein the stem cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a CD34+ hematopoietic stem cell and a hepatic stem cell.
7. The isolated cell of claim 4 , wherein the cell is selected from the group consisting of a T-cell, a B-cell, or a stem cell.
8. The isolated cell of claim 7 , wherein the stem cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a CD34+ hematopoietic stem cell, and a hepatic stem cell.
9. A kit comprising the fusion protein of claim 1 .
10. A kit comprising the polynucleotide of claim 2 .
11. A method of cleaving an endogenous HPRT gene in an isolated cell, the method comprising: transforming or transfecting said isolated cell comprising said HRPT gene with the polynucleotide of claim 2 , culturing said transformed or transfected isolated cell under conditions such that said fusion proteins is expressed and the HPRT gene is cleaved.
12. The method of claim 11 , wherein the cell is selected from the group consisting of a T-cell, a B-cell or a stem cell.
13. The method of claim 12 , wherein the stem cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a CD34+ hematopoietic stem cell, and a hepatic stem cell.
14. A method of enriching for cells modified by a nuclease at an endogenous HPRT locus, the method comprising:
cleaving said endogenous HPRT gene in said isolated cell according to the method of claim 11 ;
introducing into the cell, said polynucleotide encoding said fusion protein for modification of a second locus of the endogenous HPRT gene;
subjecting the cells to selection with 6-thioguanine, thereby enriching the cells for those in which the endogenous locus has been modified.
15. The method of claim 14 , wherein the endogenous locus is inactivated.
16. The method of claim 14 , wherein the isolated cell is selected from the group consisting of a T-cell, a B-cell or a stem cell.
17. The method of claim 16 , wherein the stem cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a CD34+ hematopoietic stem cell and a hepatic stem cell.