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 genomic integration sequence;
(b) a first tandem repeat nucleic acid consisting of 18 to 200 nucleotide base pairs;
(c) a first homing endonuclease recognition site;
(d) a target nucleic acid;
(e) a second homing endonuclease recognition site;
(f) a second tandem repeat nucleic acid consisting of 18 to 200 nucleotide base pairs; and
(g) a second genomic integration sequence,
wherein each of the first and second genomic integration sequences is of sufficient length and identity to initiate homologous recombination with a yeast genomic locus, wherein the first tandem repeat nucleic acid and the second tandem repeat nucleic acid share identical sequences, and wherein the first homing endonuclease recognition site and the second homing endonuclease recognition site share identical sequences.
2. The excisable nucleic acid construct of claim 1 , wherein each of the first and second tandem repeat nucleic acids independently consists of 18 to 150 nucleotide base pairs.
3. The excisable nucleic acid construct of claim 1 , wherein each of the first and second tandem repeat nucleic acids independently consists of 18 to 100 nucleotide base pairs.
4. The excisable nucleic acid construct of claim 1 , wherein each of the first and second homing endonuclease recognition sites independently consists of 18 to 80 nucleotide base pairs.
5. The excisable nucleic acid construct of claim 1 , wherein each of the first and second homing endonuclease recognition sites is a recognition site for a homing endonuclease selected from the group consisting of: an LAGLIDADG (SEQ ID NO: 1) homing endonuclease, a HNH homing endonuclease, an His-Cys box homing endonuclease, a GIY-YIG (SEQ ID NO: 2) homing endonuclease, and a cyanobacterial homing endonuclease.
6. The excisable nucleic acid construct of claim 1 , wherein each of the first and second homing endonuclease recognition sites is a recognition site for a homing endonuclease selected from the group consisting of: I-CreI, I-MsoI, I-SceI, I-SceIV, H-DreI, I-HmuI, I-PpoI, I-Did, I-NjaI, I-NanI, I-NitI, I-TevI, I-TevII, I-TevIII, F-TevI, F-TevII, F-CphI, PI-MgaI, I-CsmI, I-CeuI, and PI-Scel.
7. The excisable nucleic acid construct of claim 1 , wherein at least one of the first or second homing endonuclease recognition sites is a recognition site for I-SceI.
8. The excisable nucleic acid construct of claim 1 , wherein at least one of the first or second homing endonuclease recognition sites is a recognition site for F-CphI.
9. The excisable nucleic acid construct of claim 1 , wherein the target nucleic acid encodes a selectable marker.
10. The excisable nucleic acid construct of claim 9 , wherein the selectable marker is selected from the group consisting of: URA3, hygromycin B phosphotransferase, aminoglycoside phosphotransferase, zeocin resistance gene and phosphinothricin N-acetyltransferase.
11. The excisable nucleic acid construct of claim 1 , wherein each of the first and second genomic integration sequences independently consists of about 50 to 5000 nucleotide base pairs.
12. The excisable nucleic acid construct of claim 1 , wherein each of the first and second genomic integration sequences independently consists of about 500 nucleotide base pairs.
13. A yeast cell comprising the excisable nucleic acid construct of claim 1 .
14. A yeast cell comprising:
(a) an excisable nucleic acid construct comprising, in a 5′ to 3′ orientation:
(i) a first tandem repeat nucleic acid consisting of 18 to 200 nucleotide base pairs;
(ii) a first homing endonuclease recognition site;
(iii) a target nucleic acid;
(iv) a second homing endonuclease recognition site; and
(v) a second tandem repeat nucleic acid consisting of 18 to 200 nucleotide base pairs,
wherein the first tandem repeat nucleic acid and the second tandem repeat nucleic acid share identical sequences, and wherein the first homing endonuclease recognition site and the second homing endonuclease recognition site share identical sequences; and
(b) a vector comprising a homing endonuclease nucleic acid encoding a homing endonuclease capable of binding to and cleaving at or adjacent to at least one of the first or second homing endonuclease recognition sites.
15. A method of excising at least one target nucleic acid from the genome of a host cell comprising a first excisable nucleic acid construct, wherein the first excisable nucleic acid construct comprises, in a 5′ to 3′ orientation:
(a) a first tandem repeat nucleic acid consisting of 18 to 200 nucleotide base pairs;
(b) a first homing endonuclease recognition site;
(c) a first target nucleic acid;
(d) a second homing endonuclease recognition site; and
(e) a second tandem repeat nucleic acid consisting of 18 to 200 nucleotide base pairs, wherein the first tandem repeat nucleic acid and the second tandem repeat nucleic acid share identical sequences, and wherein the first homing endonuclease recognition site and the second homing endonuclease recognition site share identical sequences, wherein the method comprises contacting the first excisable nucleic acid construct with a homing endonuclease in the host cell such that the homing endonuclease cleaves at or adjacent to at least one of the first or second homing endonuclease recognition sites.
16. The method of claim 15 , wherein the host cell further comprises a second excisable nucleic acid construct, the host cell thereby comprising at least two excisable nucleic acid constructs, wherein the second excisable nucleic acid construct comprises, in a 5′ to 3′ orientation:
(a) a third tandem repeat nucleic acid;
(b) a third homing endonuclease recognition site;
(c) a second target nucleic acid;
(d) a fourth homing endonuclease recognition site; and
(e) a fourth tandem repeat nucleic acid;
wherein the third tandem repeat nucleic acid and the fourth tandem repeat nucleic acid share identical sequences, and wherein the third homing endonuclease recognition site and the fourth homing endonuclease recognition site share identical sequences, wherein the method comprises: contacting the at least two excisable nucleic acid constructs with a homing endonuclease in the host cell such that the homing endonuclease cleaves at or adjacent to at least one of the homing endonuclease recognition sites of each excisable nucleic acid construct, thereby simultaneously excising the target nucleic acids from the genome of the host cell.
17. The method of claim 16 , wherein each of the tandem repeat nucleic acids of each excisable nucleic acid construct independently consists of 18 to 200 nucleotide base pairs.
18. The method of claim 16 , wherein each of the tandem repeat nucleic acids of each excisable nucleic acid construct independently consists of 18 to 150 nucleotide base pairs.