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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 substrates3-5, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates6-9. 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.
The first step of CRISPR immune surveillance requires the processing of precursor crRNA transcripts (pre-crRNAs), consisting of repeat sequences flanking viral spacer sequences, into individual mature crRNAs that each contain a single spacer12-14. CRISPR systems use three known mechanisms to produce mature crRNAs: a dedicated endonuclease (for example, Cas6 or Cas5d in type I and III systems)15-17, coupling of a host endonuclease (for example, RNase III with a trans-activating crRNA in type II systems)18, or RNase activity intrinsic to the effector enzyme itself (for example, Cpf1 in type V systems)11.
Because type VI CRISPR loci lack an obvious Cas6- or Cas5d-like endonuclease or trans-activating crRNA10, we wondered whether C2c2 itself might possess pre-crRNA processing activity, and if so, whether the mechanism would be distinct from Cpf1, an unrelated class 2 CRISPR effector that can process pre-crRNAs11. Using purified recombinant C2c2 protein homologues from three distinct branches of the C2c2 protein family (Fig. 1a, b, Extended Data Figs 1, 2), we found that all three enzymes cleave 5'-end radiolabelled pre-crRNA substrates consisting of a full-length consensus repeat sequence and a 20-nucleotide spacer sequence (Fig. 1c). Mapping the cleavage site for each pre-crRNA-C2c2 homologue pair revealed that processing occurs either two or five nucleotides upstream of the predicted repeat-sequence hairpin structure, depending on the C2c2 homologue (Fig. 1c,...