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

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• 作者：Iason Tsarmpopoulos ; Géraldine Gourgues ; Alain Blanchard ; Sanjay Vashee ; Joerg Jores ; Carole Lartigue ; Pascal Sirand-Pugnet
• 刊名：ACS Synthetic Biology
• 出版年：2016
• 出版时间：January 15, 2016
• 年：2016
• 卷：5
• 期：1
• 页码：104-109
• 全文大小：323K
• ISSN：2161-5063

文摘

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.