Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9
| Autor: | Elleke Fenna Bosma, Koen Weenink, Richard van Kranenburg, John van der Oost, Kirsten Goijvaerts, Ioannis Mougiakos, Eric Vossen |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Streptococcus pyogenes Biomedical Engineering Bacillus homologous recombination Biology Bacillus smithii Biochemistry Genetics and Molecular Biology (miscellaneous) Microbiology 03 medical and health sciences Genome editing Microbiologie CRISPR genome editing DNA Breaks Double-Stranded Insertion bacteria Gene CRISPR/Cas9 Gene knockout VLAG Genetics Gene Editing Models Genetic Cas9 Thermophile General Medicine 030104 developmental biology CRISPR-Cas Systems Homologous recombination Research Article thermophiles |
| Zdroj: | ACS synthetic biology, 6(5), 849-861 ACS synthetic biology 6 (2017) 5 ACS Synthetic Biology |
| ISSN: | 2161-5063 |
| Popis: | Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR/Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptococcus pyogenes Cas9 (spCas9)-based genome editing to a moderate thermophile, i.e., Bacillus smithii, including a gene deletion, gene knockout via insertion of premature stop codons, and gene insertion. We show that spCas9 is inactive in vivo above 42 °C, and we employ the wide temperature growth range of B. smithii as an induction system for spCas9 expression. Homologous recombination with plasmid-borne editing templates is performed at 45-55 °C, when spCas9 is inactive. Subsequent transfer to 37 °C allows for counterselection through production of active spCas9, which introduces lethal double-stranded DNA breaks to the nonedited cells. The developed method takes 4 days with 90, 100, and 20% efficiencies for gene deletion, knockout, and insertion, respectively. The major advantage of our system is the limited requirement for genetic parts: only one plasmid, one selectable marker, and a promoter are needed, and the promoter does not need to be inducible or well-characterized. Hence, it can be easily applied for genome editing purposes in both mesophilic and thermophilic nonmodel organisms with a limited genetic toolbox and ability to grow at, or tolerate, temperatures of 37 and at or above 42 °C. |
| Databáze: | OpenAIRE |
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