Open AccessIn-Yeast Engineering of a Bacterial Genome Using CRISPR/Cas9
Author(s) -
Ιason Τsarmpopoulos,
Géraldine Gourgues,
Alain Blanchard,
Sanjay Vashee,
Joerg Jores,
Carole Lartigue,
Pascal SirandPugnet
Publication year - 2015
Publication title -
acs synthetic biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.156
H-Index - 66
ISSN - 2161-5063
DOI - 10.1021/acssynbio.5b00196
Subject(s) - biology , genome , cas9 , synthetic biology , crispr , genetics , genome engineering , mutagenesis , computational biology , mycoplasma , yeast , gene , genome editing , cloning (programming) , mutant , computer science , programming language
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.