Methods for cloning and manipulating genomes

Abstract

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.

What is claimed is: 
     
         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.