Characterizing a Novel Type II-C Anti-CRISPR, AcrIIC6

Phages are the most ubiquitous species on the planet, outnumbering bacteria ten-to-one in almost all environments. Consequently, phages exert an immense amount of pressure on bacteria to evolve defense mechanisms against phage predation. One mechanism is the adaptive immune response known as the CRISPR-Cas system. In response to the CRISPR-Cas adaptive immunity, phages have evolved small proteins called anti-CRISPRs that inhibit the CRISPR-Cas system. I characterized a novel type II-C anti-CRISPR, AcrIIC6, discovered in a prophage within Neisseria meningitidis and Pasteurella multocida. Through in vivo phage targeting, I determined that AcrIIC6Nme/Pmu inhibits Cas9 proteins that are closely related to Nme1Cas9. Furthermore, AcrIIC6Nme/Pmu only bound to sgRNA loaded Cas9 and did not affect sgRNA integrity or loading. Lastly, I showed that AcrIIC6Pmu prevents Cas9 from binding target DNA efficiently. This work shows a novel anti-CRISPR of the type II-C CRISPR-Cas system that binds to the binary Cas9:sgRNA complex and prevents Cas9 from binding DNA.M.Sc