In-Yeast Engineering of a Bacterial Genome Using CRISPR/Cas9.
ACS Synth Biol
; 5(1): 104-9, 2016 Jan 15.
Article
em En
| MEDLINE
| ID: mdl-26592087
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.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
/
Engenharia Genética
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Genoma Bacteriano
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Sistemas CRISPR-Cas
Idioma:
En
Revista:
ACS Synth Biol
Ano de publicação:
2016
Tipo de documento:
Article