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ACS Synth Biol. 2016 Jan 15;5(1):104-9. doi: 10.1021/acssynbio.5b00196. Epub 2015 Dec 7.

In-Yeast Engineering of a Bacterial Genome Using CRISPR/Cas9.

Author information

1
The J. Craig Venter Institute , 9704 Medical Center Drive, Rockville, 20850 Maryland United States .
2
International Livestock Research Institute (ILRI) , PO Box 30709, 00100 Nairobi, Kenya.
3
Institute of Veterinary Bacteriology, University of Bern , Laenggass-Stra├če 122, CH-3001 Bern, Switzerland.

Abstract

One remarkable achievement in synthetic biology was the reconstruction of mycoplasma genomes and their cloning in yeast where they can be modified using available genetic tools. Recently, CRISPR/Cas9 editing tools were developed for yeast mutagenesis. Here, we report their adaptation for the engineering of bacterial genomes cloned in yeast. A seamless deletion of the mycoplasma glycerol-3-phosphate oxidase-encoding gene (glpO) was achieved without selection in one step, using 90 nt paired oligonucleotides as templates to drive recombination. Screening of the resulting clones revealed that more than 20% contained the desired deletion. After manipulation, the overall integrity of the cloned mycoplasma genome was verified by multiplex PCR and PFGE. Finally, the edited genome was back-transplanted into a mycoplasma recipient cell. In accordance with the deletion of glpO, the mutant mycoplasma was affected in the production of H2O2. This work paves the way to high-throughput manipulation of natural or synthetic genomes in yeast.

KEYWORDS:

CRISPR/Cas9; Mycoplasma; Saccharomyces cerevisiae; genome engineering; genome transplantation; seamless gene deletion

PMID:
26592087
DOI:
10.1021/acssynbio.5b00196
[Indexed for MEDLINE]

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