Characterizing a Thermostable Cas9 and Exploring Its Potential As a Genome Engineering Tool for Both Thermophilic and Mesophilic Prokaryotes

CRISPR-Cas9 based genome engineering tools have revolutionized fundamental research and

biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature

of the already established Cas9 systems restricts their applicability, excluding thermophiles and

cellular environments in which mesophilic enzymes are easily inactivated. Here, we identify and

characterize Cas9 from the CRISPR-Cas type-IIC system of the thermophilic bacterium Geobacillus

thermodenitrificans T12 as an RNA-guided DNA endonuclease. We provide fundamental insights

into a thermophilic CRISPR-Cas family member and describe our work towards the development of

a genome editing and silencing tool readily applicable to thermophiles.