Speaker: Jack Dunkle, Assistant Professor-Department of Chemistry and Biochemistry, University of Alabama

Title: Mechanisms and specificity of Cas10 mediated interference

Abstract: CRISPR-Cas10 is a multiprotein complex that uses the sequence information in a bound crRNA to identify foreign RNA transcripts and initiate interference. Recently it was discovered that CRISPR-Cas10 synthesizes a previously unknown class of second messenger molecules upon detecting foreign transcripts, cyclic oligoadenylates (cOA) (1,2). cOA activate the Csm6 nuclease to promote RNA degradation and may also coordinate additional cellular responses. Using the S. epidermidis Cas10-Csm complex, a longstanding model for CRISPR-Cas function, we have reconstituted cOA synthesis and have found that it entails Mg2+ dependent synthesis of 3-6 nt cOAs (3). We show that activation of cOA synthesis is perturbed by single nucleotide mismatches between the crRNA and target RNA at discrete positions, and that synthesis is antagonized by Csm3-mediated target RNA cleavage. Altogether, our results establish the requirements for cOA production in a model Type III CRISPR-Cas system and suggest Csm3 mediated target RNA cutting acts as a molecular switch to shut off cOA synthesis. Since the CRISPR-Cas10 system is present in ~20% of all prokaryotic genomes sequenced (4), its role in bacteriophage defense and regulation of horizontal gene transfer are likely important to bacterial physiology in niches such as the human microbiome.