Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9.
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.
Laboratory of Microbiology, Wageningen University , Stippeneng 4, 6708 WE Wageningen, The Netherlands.
Ioannis
I
Mougiakos
Laboratory of Microbiology, Wageningen University , Stippeneng 4, 6708 WE Wageningen, The Netherlands.
Elleke F
EF
Bosma
Laboratory of Microbiology, Wageningen University , Stippeneng 4, 6708 WE Wageningen, The Netherlands.
Koen
K
Weenink
Laboratory of Microbiology, Wageningen University , Stippeneng 4, 6708 WE Wageningen, The Netherlands.
Eric
E
Vossen
Laboratory of Microbiology, Wageningen University , Stippeneng 4, 6708 WE Wageningen, The Netherlands.
Kirsten
K
Goijvaerts
Laboratory of Microbiology, Wageningen University , Stippeneng 4, 6708 WE Wageningen, The Netherlands.
John
J
van der Oost
Laboratory of Microbiology, Wageningen University , Stippeneng 4, 6708 WE Wageningen, The Netherlands.|Corbion , Arkelsedijk 46, 4206 AC Gorinchem, The Netherlands.
Richard
0000-0001-5856-1546
R
van Kranenburg
D001407:Bacillus / Q000235:genetics; D064113:CRISPR-Cas Systems / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D000072669:Gene Editing / Q000379:methods*; D008957:Models, Genetic; D013297:Streptococcus pyogenes / Q000235:genetics
Bacillus smithii; CRISPR/Cas9; bacteria; genome editing; homologous recombination; thermophiles