Regulation and specificity of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10‐Csm complex

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 have investigated the effect of target-crRNA mismatches on cOA synthesis finding that some single mismatches dramatically reduce cOA synthesis in a position dependent and sequence-context dependent manner. We have found that cOA 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. 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. 1. Kazlauskiene, M., Kostiuk, G., Venclovas, C., Tamulaitis, G., and Siksnys, V. (2017) A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science 357, 605-609 2. Niewoehner, O., Garcia-Doval, C., Rostol, J. T., Berk, C., Schwede, F., Bigler, L., Hall, J., Marraffini, L. A., and Jinek, M. (2017) Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers. Nature 548, 543-548 3. Nasef, M., Muffly, M. C., Beckman, A. B., Rowe, S. J., Walker, F. C., Hatoum-Aslan, A., and Dunkle, J. A. (2019) Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex. RNA 25, 948-962 4. Makarova, K. S., Wolf, Y. I., Alkhnbashi, O. S., Costa, F., Shah, S. A., Saunders, S. J., Barrangou, R., Brouns, S. J., Charpentier, E., Haft, D. H., Horvath, P., Moineau, S., Mojica, F. J., Terns, R. M., Terns, M. P., White, M. F., Yakunin, A. F., Garrett, R. A., van der Oost, J., Backofen, R., and Koonin, E. V. (2015) An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol 13, 722-736