Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage1,2. Although most prokaryotic adaptive immune systems generally target DNA substrates3,4,5, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates6,7,8,9. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase)9,10. How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear. Here we show that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behaviour of the evolutionarily unrelated CRISPR enzyme Cpf1 (ref. 11). The two RNase activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow sensitive detection of cellular transcripts.
We thank the QB3 MacroLab for assistance with cloning of C2c2 constructs; N. Ma and K. Zhou for technical assistance; S. N. Floor, S. C. Strutt, A. V. Wright and M. L. Hochstrasser for critical reading of the manuscript; and members of the Doudna, Cate and Tjian laboratories for discussions. S.C.K. acknowledges support from the National Science Foundation Graduate Research Fellowship Program; M.R.O. is a recipient of a C. J. Martin Overseas Early Career Fellowship from the National Health and Medical Research Council of Australia. This work was supported in part by a Frontiers Science award from the Paul Allen Institute to J.A.D., the National Science Foundation (MCB-1244557 to J.A.D.), the California Institute for Regenerative Medicine (CIRM, RB4-06016 to R.T.), and the National Institutes of Health (P50-GM102706 to J.H.D.C). R.T. and J.A.D. are Investigators of the Howard Hughes Medical Institute. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine and Intellia Therapeutics and a scientific advisor to Caribou, Intellia, eFFECTOR Therapeutics, and Driver. A.E.S., M.R.O., S.C.K., J.H.D.C. and J.A.D. have filed a patent application related to this work.
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This file contains source data BAM files for sequencing reanalysis.