A Thermophilic Cas9 Specific for Tandem Cytosine-Adjacent DNA and Sensitive to DNA Topology

Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the Cas9-bound guide RNA and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition in time and space of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from thermophile Acidothermus cellulolyticus (AceCas9). Acidothermus cellulolyticus has been used for producing the enzyme endo-1, 4-β-glucanase (E1) which is used for the commercial hydrolysis of cellulose into glucose. Genome editing in A. cellulylyticus could impact its utilization in bioenergy development. We found that AceCas9 depends strictly on a 5’-NNNCC-3’ PAM and is more efficient in cleaving negative supercoils than relaxed DNA. We further characterized the dependence of AceCas9 on temperature, guide length, divalent metal ions, and mismatches to the guide RNA. The thermostability, cytosine-specific and DNA topology-sensitive properties of the AceCas9 maybe explored for specific genome editing applications.