Structural insights into the CRISPR-Cas-associated ribonuclease activity of Staphylococcus epidermidis Csm3 and Csm6

Abstract The C lustered R egularly I nterspaced S hort P alindromic R epeats (CRISPR)- C RISPR- as sociated (Cas) system is an adaptive immune system in bacteria and archaea that resists exogenous invasion through nucleic acid-mediated cleavage. In the type III-A system, the Csm complex contains five effectors and a CRISPR RNA, which edits both single stranded RNA and double stranded DNA. It has recently been demonstrated that cyclic oligoadenylates (cOAs), which are synthesized by the Csm complex, act as second messengers that bind and activate Csm6. Here, we report the crystal structures of Staphylococcus epidermidis Csm3 (SeCsm3) and an N-terminally truncated Csm6 (SeCsm6ΔN) at 2.26 and 2.0 A, respectively. The structure of SeCsm3 highly resembled previously reported Csm3 structures from other species; however, it provided novel observations allowing further enzyme characterization. The homodimeric SeCsm6ΔN folds into a compact structure. The dimerization of the HEPN domain leads to the formation of the ribonuclease active site, which is consistent with the reported Csm6 structures. Altogether, our studies provide a structural view of the ribonuclease activity mediated by Csm3 and Csm6 of the type III-A CRISPR-Cas system.