Methods for cloning and manipulating genomes
Compositions and methods are disclosed herein for cloning a synthetic or a semi-synthetic donor genome in a heterologous host cell. In one embodiment, the donor genome can be further modified within a host cell. Modified or unmodified genomes can be further isolated from the host cell and transferred to a recipient cell. Methods disclosed herein can be used to alter donor genomes from intractable donor cells in more tractable host cells.
1. A method for seamlessly introducing a modification in a target nucleic acid molecule present in a host cell, comprising:
a. introducing a mutagenesis construct and a host vector into the host cell whereby the host vector recombines with the mutagenesis construct in the host cell,
wherein the mutagenesis construct contains
a first portion of homology that is homologous to a sequence of the target nucleic acid molecule upstream or downstream of a target gene to be modified in the target region on the target nucleic acid molecule, wherein upon recombination the first portion of homology generates a tandem repeat with the homologous sequence of the target nucleic acid molecule;
a second portion of homology that is homologous to a 5′ portion of the target region on the target nucleic acid molecule upstream of the modification;
a gene encoding an endonuclease having an inducible promoter;
an endonuclease recognition site for the encoded endonuclease; and
a selectable marker;
a third portion of homology that is homologous to a 3′ portion of the target region on the target nucleic acid molecule downstream of the modification;
wherein the second and/or third portions of homology comprise one or more nucleotide modifications compared to the homologous portion in the target nucleic acid; and
b. incubating the cells under conditions whereby the mutagenesis construct is integrated into the target nucleic acid molecule and tandem repeats are generated that flank the target region on the target nucleic acid molecule between the first portion of homology and a sequence upstream or downstream of the target region; and
inducing the inducible promoter, whereby the endonuclease is expressed and promotes one or more double-strand break cleavages in the target nucleic acid molecule;
i. whereby recombination occurs between the first portion of homology and the upstream or downstream portion of the target region on the target nucleic acid molecule that is homologous to the first portion of homology, thereby seamlessly removing a portion of the construct;
whereby a modification is seamlessly introduced into the target nucleic acid molecule.
2. The method of claim 1 wherein the endonuclease recognition site is an I-SceI recognition site.
3. The method of claim 1 wherein the promoter is a GAL-1 promoter.
4. The method of claim 1 wherein the selectable marker is URA3.
5. The method of claim 1 wherein the endonuclease recognition site is an I-SceI recognition site, the promoter is a GAL-1 promoter, and the selectable marker is URA3.
6. The method of claim 1 wherein the portion of the construct that is removed is the selectable marker.
7. The method of claim 1 wherein the tandem repeats that flank the target region flank the selectable marker.
8. The method of claim 1 wherein the host cell is a yeast cell.
9. The method of claim 8 wherein the yeast cell is Saccharomyces cerevisiae or Saccharomyces pombe.
10. The method of claim 8 wherein the target nucleic acid molecule is a bacterial, cyanobacterial, or microalgal donor genome.
11. The method of claim 10 wherein the target nucleic acid molecule is a bacterial genome.
12. The method of claim 11 wherein the target nucleic acid molecule is a Mycoplasma genome.
13. The method of claim 8 wherein the modification is introduced by a homologous recombination.
14. The method of claim 12 wherein the modification is introduced by a homologous recombination.
15. The method of claim 13 wherein the modification is selected from the group consisting of: a point mutation, a substitution, an insertion, and the modification of a nucleotide.
16. The method of claim 14 wherein the modification is selected from the group consisting of: a point mutation, a substitution, an insertion, and the modification of a nucleotide.
17. The method of claim 1 wherein:
a. the host cell is a yeast of the genus Saccharomyces;
b. the endonuclease recognition site is a I-SceI recognition site;
c. the promoter is a GAL-1 promoter; and
d. the selectable marker is URA3.
18. The method of claim 1 wherein the second and third portions of homology flank the first portion of homology.