Nucleic acids, compositions and methods for the excision of target nucleic acids
View Patent ↗Nucleic acids, compositions, and methods that allow for the excision of one or more loci from the genome of a host cell are provided herein. In particular, provided herein is an excisable nucleic acid construct comprising, in a 5′ to 3′ orientation: a first tandem repeat nucleic acid, a first homing endonuclease recognition site, a target nucleic acid, a second homing endonuclease recognition site, and a second tandem repeat nucleic acid. In some embodiments, the excisable nucleic acid construct is integrated into the host cell genome, and the target nucleic acid can be excised from the host cell genome by contacting the homing endonuclease recognition sites with one or more appropriate homing endonucleases.
1. An excisable nucleic acid construct comprising, in a 5′ to 3′ orientation:
(a) a first tandem repeat nucleic acid;
(b) a first F-CphI endonuclease recognition site;
(c) a target nucleic acid;
(d) a second F-CphI endonuclease recognition site; and
(e) a second tandem repeat nucleic acid.
2. The excisable nucleic acid construct of claim 1 , wherein each of the first and second tandem repeat nucleic acids independently comprises 18-80 nucleotide base pairs.
3. The excisable nucleic acid construct of claim 1 , wherein the target nucleic acid encodes a selectable marker.
4. The excisable nucleic acid construct of claim 3 , wherein the selectable marker is selected from the group consisting of: URA3, hygromycin B phosphotransferase, aminoglycoside phosphotransferase, zeocin resistance gene and phosphinothricin N-acetyltransferase.
5. The excisable nucleic acid construct of claim 1 , further comprising a first integration site linked 5′ of the first homing endonuclease recognition site and a second integration site linked 3′ of the second tandem repeat nucleic acid.
6. The excisable nucleic acid construct of claim 1 , wherein the target nucleic acid comprises a promoter element operably linked to a nucleic acid encoding F-CphI endonuclease.
7. A host cell comprising:
(a) the excisable nucleic acid construct of claim 1 ; and
(b) a vector comprising a nucleic acid encoding F-CphI endonuclease.
8. The host cell of claim 7 , wherein the vector comprises a promoter element that controls the expression of the nucleic acid encoding F-CphI endonuclease.
9. The host cell of claim 8 , wherein the promoter element is an inducible promoter.
10. The host cell of claim 7 that is a yeast cell.
11. The host cell of claim 10 that is a haploid yeast cell.
12. The host cell of claim 10 that is a diploid yeast cell.
13. The host cell of claim 10 that is a Saccharomyces cerevisiae cell.
14. The host cell of claim 7 , wherein the excisable nucleic acid construct is integrated into the host cell genome.
15. A method of excising at least one target nucleic acid from the genome of a host cell comprising the excisable nucleic acid construct of claim 1 , wherein the method comprises: contacting the excisable nucleic acid construct with F-CphI in the host cell.
16. The method of claim 15 , wherein said excising operably links a promoter element to a gene of interest.
17. The method of claim 15 , wherein the host cell is a yeast cell.
18. The method of claim 15 , wherein the host cell is a haploid yeast cell.
19. The method of claim 15 , wherein the host cell is a diploid yeast cell.
20. The method of claim 15 , wherein the host cell is a Saccharomyces cerevisiae cell.