Methods and compositions for targeted integration
Disclosed herein are methods and compositions for targeted integration of one or more copies of a sequence of interest using zinc finger nucleases (ZFNs) comprising a zinc finger protein and a cleavage domain or cleavage half-domain and integrase defective lentiviral donor constructs.
1. An integrase-defective lentiviral (IDLV) donor polynucleotide comprising, between the lentivirus LTRs,
a gene of interest and first and second nucleotide sequences that are homologous sequences to a region of interest of endogenous cellular chromatin of a mammalian cell, wherein the homologous sequences are identical to endogenous cellular chromatin sequences of the mammalian cell; and further wherein the donor polynucleotide is selected from the group consisting of:
(i) a donor polynucleotide comprising the gene of interest flanked by the first and second homologous sequences and a selectable marker outside the first and second homologous sequences;
(ii) a donor polynucleotide comprising the gene of interest, a first selectable marker, and a second selectable marker, wherein the gene or interest and the first selectable marker are flanked by the first and second homologous sequences and further wherein the second selectable marker is outside the first and second homologous sequences;
(iii) a donor polynucleotide comprising the gene of interest and a selectable marker, wherein the gene of interest and the selectable marker are outside the first and second homologous sequences; and
(iv) a donor polynucleotide comprising the gene of interest and a selectable marker, wherein the gene of interest and the selectable marker are flanked by the first and second homologous sequences.
2. The IDLV donor polynucleotide of claim 1 , wherein the first or second selectable marker is a positive selection marker.
3. The IDLV donor polynucleotide of claim 1 , wherein the first or second selectable marker is a negative selection marker.
4. The IDLV donor polynucleotide of claim 1 , wherein the first selectable marker is a positive selection marker and the second selectable marker is a negative selection marker.
5. The IDLV donor polynucleotide of claim 4 , wherein the positive-negative selection marker is hyg-TK.
6. The IDLY donor polynucleotide of claim 1 , wherein the cell is a human cell.
7. The DLV donor polynucleotide of claim 1 , wherein the first and second nucleotides flank the exogenous sequence.
8. A method for controlled, site-specific integration of a single copy or multiple copies of an exogenous nucleic acid sequence into a cell, the method comprising:
(a) expressing a first fusion protein in the cell, the first fusion protein comprising a first zinc finger binding domain and a first cleavage half-domain, wherein the first zinc finger binding domain has been engineered to bind to a first target site in a region of interest in the genome of the cell;
(b) expressing a second fusion protein in the cell, the second fusion protein comprising a second zinc finger binding domain and a second cleavage half domain, wherein the second zinc finger binding domain binds to a second target site in the region of interest in the genome of the cell, wherein the second target site is different from the first target site; and
(c) contacting the cell with any of the IDLV donor polynucleotides of claim 1 ;
wherein binding of the first fusion protein to the first target site, and binding of the second fusion protein to the second target site, positions the cleavage half-domains such that the genome of the cell is cleaved in the region of interest, thereby resulting in integration of one or more copies of the exogenous sequence into the genome of the cell in the region of interest.
9. The method according to claim 8 , wherein the exogenous nucleic acid sequence comprises a cDNA.
10. The method of claim 8 , wherein the region of interest is in a region of the genome that is not essential for viability.
11. The method of any claim 8 , wherein the region of interest is in a region of the genome that is transcriptionally active.
12. The method of claim 8 , wherein a single copy of the exogenous sequence is integrated into the genome of the cell.
13. The method of claim 8 , wherein multiple copies of the exogenous sequence are integrated into the genome of the cell.
14. The method according to claim 8 , wherein at least one of the fusion proteins comprises an alteration in the amino acid sequence of the dimerization interface of the cleavage half-domain.
15. The method according to claim 8 , wherein the cell is a mammalian cell.
16. The method according to 15 , wherein the cell is a human cell.