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

The paper report the reconstruction of mycoplasma genome 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 and reduced-cost manipulation of natural or synthetic genomes in yeast. Moreover, this work will foster the development of CRISPR/Cas9-based editing tools that can be directly applied to Mycoplasma.