Cas10 residues lining the target RNA binding channel regulate interference by distinguishing cognate target RNA from mismatched targets

Type III CRISPR systems are defined by the presence of the Cas10 protein and are among the most abundant CRISPR systems in nature. Cas10 forms a complex with crRNA and several Cas proteins that surveils bacterial cells for foreign RNA molecules and when they are detected it activates a cascade of interference activities. The synthesis of the cyclic oligoadenylate signaling molecule by Cas10 is a key aspect of the interference cascade. Despite structures of the Cas10 complex bound to target RNAs, the molecular mechanism by which Cas10 senses the bound state to license interference is lacking. We identified five residues in S. epidermidis Cas10, two in the Cas10 Palm2 domain and three in domain 4, that line the target RNA binding channel. We assessed the contribution of these residues to interference in the context of a cognate or mismatched target RNA. We found that the residues regulate whether a mismatched crRNA-target RNA duplex is able to activate interference in vivo. We purified two site-directed mutants of Cas10-Csm and show with in vitro cOA synthesis assays they demonstrate enhanced discrimination of cognate versus mismatched targets.