METHODS FOR CLONING AND MANIPULATING GENOMES
Compositions and methods are disclosed herein for cloning a 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 to a 5′ portion of the target nucleic acid molecule upstream of the modification;
an endonuclease recognition site;
a promoter;
a gene encoding the endonuclease; and
a selectable marker;
a second repeat portion of homology that is homologous to a sequence of the target nucleic acid molecule upstream of a target region on the target nucleic acid molecule;
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; and
b. incubating the cells under conditions
i. whereby recombination occurs between the first portion of homology and the upstream or downstream portion, thereby seamlessly removing a portion of the construct,
ii. that promote one or more double-strand break cleavages in the target nucleic acid molecule near the target region containing 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 6 wherein the endonuclease is inducibly expressed.
8 . The method of claim 7 wherein the double strand break cleavage is generated by the inducibly expressed endonuclease.
9 . The method of claim 8 further comprising the introduction of a sequence generating tandem repeat regions that flank the target region or portion thereof on the target nucleic acid molecule.
10 . The method of claim 9 wherein the tandem repeat regions that flank the target region or portion thereof flank the selectable marker.
11 . The method of claim 1 wherein the host cell is a yeast cell.
12 . The method of claim 11 wherein the yeast cell is Saccharomyces cerevisiae or Saccharomyces pombe.
13 . The method of claim 11 wherein the target nucleic acid molecule is a bacterial, cyanobacterial, or microalgal donor genome.
14 . The method of claim 13 wherein the target nucleic acid molecule is a bacterial genome.
15 . The method of claim 14 wherein the target nucleic acid molecule is a Mycoplasma genome.
16 . The method of claim 11 wherein the modification is a homologous recombination.
17 . The method of claim 15 wherein the modification is a homologous recombination.
18 . The method of claim 16 wherein the homologous recombination is selected from the group consisting of: a point mutation, a substitution, an insertion, and the modification of a nucleotide.
19 . The method of claim 17 wherein the homologous recombination is selected from the group consisting of: a point mutation, a substitution, an insertion, and the modification of a nucleotide.
20 . 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;
d. the selectable marker is URA3; and
e. the endonuclease is inducibly expressed and the double strand break cleavage is generated by the inducibly expressed endonuclease.