Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9

Ioannis Mougiakos, Elleke Fenna Bosma, Koen Weenink, Eric Vossen, Kirsten Goijvaerts, John van der Oost, Richard van Kranenburg

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Abstract

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.
Original languageEnglish
JournalA C S Synthetic Biology
Volume6
Issue number5
Pages (from-to)849-861
ISSN2161-5063
DOIs
Publication statusPublished - 2017
Externally publishedYes

Bibliographical note

This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes.

Keywords

  • CRISPR/Cas9
  • Bacteria
  • Bacillus smithii
  • Thermophiles
  • Genome editing
  • Homologous recombination

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