Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

By Alexandra East-Seletsky, M. O’Connell, Spencer C. Knight, D. Burstein, J. Cate, R. Tjian, Jennifer A. Doudna. Nature, 2016

1,064 citations recorded by the source corpus.

Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). 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.

Subfields: Endocrinology, Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/00c259e27c56b5d745b782202181828ab14e589f

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Citations in the corpus

Nucleic acid detection with CRISPR-Cas13a/C2c2 (2017, positive): “Although this sensitivity is an improvement on previous studies with LbCas13a (12), attomolar sensitivity is required for many diagnostic applications (14–16).”
CRISPR technology: A decade of genome editing is only the beginning (2023, background): “Some of these enzymes have also been harnessed for the development of imaging methods (18–20) and diagnostic approaches (21, 22).”
Transcriptome engineering with RNA-targeting Type VI-D CRISPR effectors (2018, positive): “…et al., 2017; Samai et al., 2015), type VI systems have been recently described as the only known single-effector CRISPR nucleases that exclusively target RNA (Abudayyeh et al., 2016; East-Seletsky et al., 2016; Konermann et al., 2018; Shmakov et al., 2015; Smargon et al., 2017; Yan et al., 2018).”
The Biology of CRISPR-Cas: Backward and Forward. (2018, positive): “In contrast to the internal active sites of other class 2 effectors (Dong et al., 2016; Jinek et al., 2014; Yang et al., 2016), the two HEPN domains of Cas13a form a composite active site at the external surface of the enzyme that is proposed to account for non-speciﬁc RNA degradation (Liu et al., 2”
RNA virus interference via CRISPR/Cas13a system in plants (2017, background): “Importantly in planta, the absence of the processed mature crRNA product in the wild-type plants, in contrast to pCas13a-OE plants, provides corroborating evidence that the generation of the mature crRNA is attributed to the presence of pCas13a, consistent with the known role of Cas13a in processing”
Field-deployable viral diagnostics using CRISPR-Cas13 (2018, background): “Cas13, an RNA-guided ribonuclease, provides specificity through CRISPR RNA (crRNA):target pairing, and additional sensitivity due to signal amplification by Cas13’s collateral cleavage activity(14, 15).”
Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy (2020, neutral): “Cas12 and Cas13 proteins are RNA-guided components of bacterial adaptive immune systems that directly target single- and double-stranded DNA or single-stranded (ss) RNA substrates, respectively (Abudayyeh et al., 2016; Chen et al., 2018; East-Seletsky et al., 2016; Zetsche et al., 2015).”