Abstract 1329 Structural and biochemical analysis of a Type III-A CRISPR-Cas system supports the idea of a Cas10-activating region

Prokaryotes rely on various defense mechanisms to protect themselves against bacteriophages. One type of these systems is the Type III CRISPR-Cas system, a multiprotein effector complex that provides adaptive immunity. The effector complex is bound to a strand of CRISPR RNA (crRNA) to aid in the detection of viral transcripts. The system of interest is CRISPR-Cas10. In this specific complex, binding of a viral transcript to the crRNA activates the effector complex, initiating a cascade of events to protect the cell. The signature protein, Cas10, modulates the synthesis of cyclic oligoadenylates (cOAs), second messenger molecules, which activate nucleotide cleavage by the ancillary protein Csm6 to destroy the viral hereditary material. It is thought the crRNA base pairs with the viral target in a localized segment of the Cas10 protein to form a Cas10-activating region (CAR). Complementarity along the crRNA-target duplex near the CAR region is thought to have a larger affect on cOA synthesis compared to other regions on the duplex. Through cryo-EM, the structure of the S. epidermidis Cas10-Csm complex bound to target RNA was elucidated, establishing a molecular model encompassing four of the five proteins and both RNAs. This structural insight reveals how the complex interacts with both crRNA and target RNA. To complement this structural analysis, in vitro and in vivo assays were conducted to evaluate the impact of mismatched base pairs on cOA synthesis. These results strongly support the existence of a CAR in type III-A CRISPR demonstrating a strong correlation between duplex complementarity along the CAR and cOA production. Collectively, the structural and biochemical data contribute to a better understanding of the defense mechanism in bacteria which harbor these type III CRISPR systems. Additionally, this knowledge holds significance for the dynamic development of nucleic acid diagnostics (CRISPR-Dx). This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health (Award Number R35GM142966).