Project Summary CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins are a group of diverse crRNA guided nucleases providing prokaryotes adaptive immunity to foreign genetic elements. Cas10-Csm is an approximately 300 kDa multiprotein complex that upon detecting foreign RNA transcripts initiates a DNA and RNA degradation response. Distinct CRISPR-Cas types use the information in crRNA to detect either double-stranded DNA or single-stranded RNA. While much is known concerning the specificity mechanisms of dsDNA detection by Type I (Cascade) and Type II (Cas9) systems, little is known concerning the specificity mechanisms of ssRNA detection by Type III (Cas10) systems. This is significant because evidence exists that the mechanisms for enforcing specificity during dsDNA detection and ssRNA detection are fundamentally different: competition between rehybridization of the non-base paired DNA strand to re-form dsDNA or binding to a Cas protein is essential to the dsDNA detecting specificity mechanism. This mechanism is not possible during specific detection of ssRNA. The hypothesis will be tested that in the Cas10- Csm system mismatches in specific, sensitive locations within the crRNA-target RNA duplex disrupt interference. Cas10-Csm structural models suggest the Csm2 component of Cas10-Csm directly contacts bound target RNA. We will test the hypothesis that Csm2 plays an integral role in detecting cognate RNA binding to Cas10-Csm and relays this signal to Cas10 via structural changes. We will use cryo-EM to determine the `structural mechanism' for Cas10 activation. The research proposed will impact several emerging biotechnologies such as the deployment of Cas10 as a point-of-care RNA virus diagnostic.