Reconstruction of site specific nuclease binding sites
Disclosed herein are methods and compositions for the repair of site specific nuclease binding sites by targeted integration and/or targeted excision of one or more sequences into a cell.
1. A method for producing a repaired site specific nuclease binding site within a plant, the method comprising the following steps:
a. Providing a plant genome comprising a first copy of a site specific nuclease binding site, an intervening polynucleotide sequence, and a second copy of the site specific nuclease binding site, wherein the first copy of the site specific nuclease binding site and the second copy of the site specific nuclease binding site are identical, and the first copy of the site specific nuclease binding site is located within 3,000 bp to 4,000 bp of the second copy of the site specific nuclease binding site;
b. Introducing a site specific nuclease designed to bind and cleave at the site specific nuclease binding site;
c. Cleaving the first copy of the site specific nuclease binding site;
d. Cleaving the second copy of the site specific nuclease binding site;
e. Excising the intervening polynucleotide sequence from the plant genome;
f. Recombining the first cleaved site specific nuclease binding site with the second cleaved site specific nuclease binding site;
g. Producing the repaired site specific nuclease binding site, wherein the repaired site specific nuclease binding site is repaired via a NHEJ-mediated cellular process and the repaired site specific nuclease binding site is identical to the first copy of the site specific nuclease binding site.
2. The method of claim 1 , wherein the site specific nuclease is selected from the group consisting of a Zinc Finger nuclease, a CRISPR nuclease, a TALEN nuclease, or any combination thereof.
3. The method of claim 2 , wherein the Zinc Finger nuclease comprise a FokI nuclease.
4. The method of claim 3 , wherein the FokI nuclease comprises a high fidelity FokI nuclease.
5. The method of claim 1 , wherein the intervening polynucleotide sequence is completely removed from the plant genome.
6. The method of claim 5 , wherein the intervening polynucleotide sequence comprises a transgene.
7. The method of claim 6 , wherein the transgene encodes a selectable marker.
8. The method of claim 1 , wherein the repaired site specific nuclease binding site is greater than 6 bp in length.
9. The method of claim 1 , wherein the repaired site specific nuclease does not comprise an INDEL.
10. The method of claim 1 , wherein the first copy of the site specific nuclease binding site, the intervening polynucleotide sequence, and the second copy of the site specific nuclease comprise either a native plant genomic sequence or a transgenic sequence within a plant genome.
11. The method of claim 1 , the method further comprising the steps of:
h. Targeting the repaired site specific nuclease binding site with the site specific nuclease;
i. Cleaving the repaired site specific nuclease binding site with the site specific nuclease;
j. Introducing a donor polynucleotide sequence;
k. Integrating the donor polynucleotide sequence within the cleaved site specific nuclease binding site; and
l. Producing a plant genome comprising the donor polynucleotide sequence stably integrated within the plant genome.
12. The method of claim 1 , wherein the intervening polynucleotide sequence comprises a polynucleotide encoding a transgene or a gene expression cassette.
13. The method of claim 1 , wherein the site specific nuclease binding site is palindromic.
14. The method of claim 1 , wherein the site specific nuclease binding site is non-palindromic.
15. The method of claim 1 , wherein the repaired site specific nuclease binding site is inherited in a progeny.