Characterizing a thermostable Cas9 for bacterial genome editing and silencing

Ioannis Mougiakos, Prarthana Mohanraju, Elleke F. Bosma, Valentijn Vrouwe, Max Finger Bou, Mihris I. S. Naduthodi, Alex Gussak, Rudolf B. L. Brinkman, Richard Kranenburg and John Oost ()
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Ioannis Mougiakos: Wageningen University
Prarthana Mohanraju: Wageningen University
Elleke F. Bosma: Wageningen University
Valentijn Vrouwe: Wageningen University
Max Finger Bou: Wageningen University
Mihris I. S. Naduthodi: Wageningen University
Alex Gussak: Wageningen University
Rudolf B. L. Brinkman: Corbion
Richard Kranenburg: Wageningen University
John Oost: Wageningen University

Nature Communications, 2017, vol. 8, issue 1, 1-11

Abstract: Abstract CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

Date: 2017
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DOI: 10.1038/s41467-017-01591-4

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