title: In-Yeast Engineering of a Bacterial Genome Using CRISPR/Cas9.
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.
authors.forename: Iason
authors.initials: I
authors.lastname: Tsarmpopoulos
authors.forename: Géraldine
authors.initials: G
authors.lastname: Gourgues
authors.forename: Alain
authors.initials: A
authors.lastname: Blanchard
authors.affiliation: The J. Craig Venter Institute , 9704 Medical Center Drive, Rockville, 20850 Maryland United States .
authors.forename: Sanjay
authors.initials: S
authors.lastname: Vashee
authors.affiliation: International Livestock Research Institute (ILRI) , PO Box 30709, 00100 Nairobi, Kenya.|Institute of Veterinary Bacteriology, University of Bern , Laenggass-Straße 122, CH-3001 Bern, Switzerland.
authors.forename: Joerg
authors.initials: J
authors.lastname: Jores
authors.forename: Carole
authors.initials: C
authors.lastname: Lartigue
authors.forename: Pascal
authors.initials: P
authors.lastname: Sirand-Pugnet
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D017353:Gene Deletion; D005800:Genes, Fungal; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000378:metabolism; D016680:Genome, Bacterial; D009174:Mycoplasma / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D012331:RNA, Fungal
keywords: CRISPR/Cas9; Mycoplasma; Saccharomyces cerevisiae; genome engineering; genome transplantation; seamless gene deletion