Tandem repeat coupled with endonuclease cleavage (TREC): a seamless modification tool for genome engineering in yeast.
The complete synthetic Mycoplasma genitalium genome ( approximately 583 kb) has been assembled and cloned as a circular plasmid in the yeast Saccharomyces cerevisiae. Attempts to engineer the cloned genome by standard genetic methods involving the URA3/5-fluoroorotic acid (5-FOA) counter-selection have shown a high background of 5-FOA resistant clones derived from spontaneous deletions of the bacterial genome maintained in yeast. Here, we report a method that can seamlessly modify the bacterial genome in yeast with high efficiency. This method requires two sequential homologous recombination events. First, the target region is replaced with a mutagenesis cassette that consists of a knock-out CORE (an18-bp I-SceI recognition site, the SCEI gene under the control of the GAL1 promoter, and the URA3 marker) and a DNA fragment homologous to the sequence upstream of the target site. The replacement generates tandem repeat sequences flanking the CORE. Second, galactose induces the expression of I-SceI, which generates a double-strand break (DSB) at the recognition site. This DSB promotes intra-molecular homologous recombination between the repeat sequences, and leads to an excision of the CORE. As a result, a seamless modification is generated. This method can be adapted for a variety of genomic modifications and may provide an important tool to modify and design natural or synthetic genomes propagated in yeast.
The J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA. vnoskov@jcvi.org
Vladimir N
VN
Noskov
Thomas H
TH
Segall-Shapiro
Ray-Yuan
RY
Chuang
D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D045704:Mycoplasma genitalium / Q000235:genetics*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D020080:Tandem Repeat Sequences
D029701:Saccharomyces cerevisiae Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific