Targeted integration into the PPP1R12C locus
Disclosed herein are methods and compositions for targeted integration of an exogenous sequence into the human PPP1R12C locus, for example, for expression of a polypeptide of interest.
1. A composition comprising:
one or more polynucleotides encoding one or more nucleases, each nuclease comprising (i) a DNA-binding domain and (ii) a cleavage domain or cleavage half-domain, wherein the DNA-binding domain has been engineered to bind to a first target site in intron 1 of a PPP1R12C gene in the genome of an isolated cell.
2. The composition of claim 1 , wherein the one or more polynucleotides encode at least two nucleases.
3. The composition of claim 1 , further comprising an isolated nucleic acid sequence.
4. The composition of claim 3 , wherein the isolated nucleic acid sequence encodes a polypeptide.
5. The composition according to claim 4 , wherein the polypeptide is selected from the group consisting of an antibody, an antigen, an enzyme, a growth factor, a cell surface receptor, a nuclear receptor, a hormone, a lymphokine, a cytokine, a reporter, functional fragments thereof and combinations thereof.
6. The composition of claim 5 , wherein the reporter comprises GFP.
7. The composition of claim 3 , wherein the polynucleotide is selected from the group consisting of one or more shRNAs, one or more RNAi molecules, one or more miRNAs and combinations thereof.
8. The composition of claim 3 , wherein the isolate nucleic acid sequence further comprises a promoter.
9. The composition of claim 3 , wherein the isolated nucleic acid sequence does not comprise a promoter.
10. The composition of claim 3 , wherein the isolated nucleic acid sequence further comprises a first nucleotide sequence that is homologous but non-identical to a first sequence in the PPP1R12C gene.
11. The composition of claim 10 , wherein the isolated nucleic acid sequence further comprises a second nucleotide sequence that is homologous but non-identical to a second sequence in the PPP1R12C gene.
12. The composition of claim 10 , wherein the isolated nucleic acid sequence comprises a tandem cassette.
13. The composition of claim 3 , wherein the isolated nucleic acid sequence is a plasmid.
14. The composition of claim 3 , wherein the isolated nucleic acid sequence is a linear DNA molecule.
15. The composition of claim 1 , wherein the cleavage half-domains are from a Type IIS restriction endonuclease.
16. The composition of claim 15 , wherein the Type IIS restriction endonuclease is selected from the group consisting of FokI and StsI.
17. The composition of claim 1 , wherein at least one of the cleavage half-domains comprises an alteration in the amino acid sequence of a dimerization interface of the cleavage half-domain.
18. The composition of claim 1 , wherein the polynucleotide is within an isolated cell.
19. The composition of claim 18 , wherein the cell is a stem cell.
20. The composition of claim 19 , wherein the stem cell is a hematopoietic stem cell.