TSARMPOPOULOS, Iason; GOURGUES, Geraldine; BLANCHARD, Alain; VASHEE, Sanjay; JORES, Joerg; LARTIGUE, Carole; SIRAND-PUGNET, Pascal

doi:10.1021/acssynbio.5b00196

Métadonnées

Afficher la notice complète

Licence d’utilisation du document

TSARMPOPOULOS, Iason
Biologie du fruit et pathologie [BFP]

GOURGUES, Geraldine
Biologie du fruit et pathologie [BFP]

BLANCHARD, Alain
Biologie du fruit et pathologie [BFP]

Langue

Article de revue

Ce document a été publié dans

ACS Synthetic Biology. 2016, vol. 5, n° 1, p. 104-109

American Chemical Society

Résumé en anglais

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.< Réduire

Mots clés

CRISPR/Cas9

Saccharomyces cerevisiae

genome transplantation

Mots clés en anglais

Mycoplasma

genome engineering

seamless gene deletion