Reference: Tsarmpopoulos I, et al. (2016) In-Yeast Engineering of a Bacterial Genome Using CRISPR/Cas9. ACS Synth Biol 5(1):104-9

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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.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, Non-P.H.S.
Authors
Tsarmpopoulos I, Gourgues G, Blanchard A, Vashee S, Jores J, Lartigue C, Sirand-Pugnet P
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