Líffræðifélag Íslands - biologia.is
Líffræðiráðstefnan 2017

Erindi/veggspjald / Talk/poster E18

Thermostable sgRNA-guided Cas9 nuclease system with novel PAM specificity

Höfundar / Authors: Björn Þór Aðalsteinsson (1), William Merre (2), Ólafur H. Friðjónsson (1), Guðmundur Hreggviðsson (1,3)

Starfsvettvangur / Affiliations: 1. Matís,2. Polytech Marseille, Aix-Marseille Université (Marseille, Frakkland), 3. Háskóli Íslands

Kynnir / Presenter: Björn Þór Aðalsteinsson

Objectives: We aimed to identify a thermostable RNA guided Cas9 nuclease system and demonstrate its activity in vitro.
Results: A type II CRISPR-Cas9 system was identified in the thermophilic bacterium Geobacillus LC300. The system’s Cas9 nuclease (gbCas9) was expressed in E coli, and a single guide RNA (sgRNA) with a 30 bp spacer sequence was designed. It was demonstrated that the sgRNA guides gbCas9 to sequences programmed in its spacer sequence, resulting in DNA double strand break – hallmarks of CRISPR-Cas9 systems. It was shown that gbCas9 is active at temperatures above to 60°C, making it suitable for in vivo adaptation for genome engineering of thermophilic bacteria. Finally, the sequence specificity of gbCas9, the protospacer adjacent motif, was found to include a GAAA motif, but is likely to include some additional sequence.
Conclusions: We demonstrate a thermostable sgRNA-guided Cas9 nuclease system active at temperatures over 60°C in vitro.