Journal Article

Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection

Knight, Spencer C., O’Connell, Mitchell R., Cate, Jamie H. D., East-Seletsky, Alexandra, Burstein, David, Tjian, Robert, Doudna, Jennifer A.

2016

Overview

The CRISPR-associated bacterial enzyme C2c2 is shown to contain two separable, distinct sites for the highly sensitive detection and cleavage of single-stranded RNA. The RNA cleaving enzyme C2c2 The programmed sequence-specific cleavage of RNA and DNA by CRISPR-associated enzymes has revolutionized genome editing. An alternative to canonical Cas9 nuclease, C2c2, was recently described. Jennifer Doudna and colleagues have probed the biochemistry of this enzyme further, and find that it contains two separable distinct sites that catalyse RNA cleavage. The authors exploit the properties of the second site to show that the enzyme can be used for highly sensitive detection and cleavage of single-stranded RNA. Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage 1 , 2 . Although most prokaryotic adaptive immune systems generally target DNA substrates 3 , 4 , 5 , type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates 6 , 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/326/2521 / 631/337/1645 / 631/45/500 / 631/45/607/1164 / Base Sequence / Clustered Regularly Interspaced Short Palindromic Repeats - genetics / CRISPR-Associated Proteins - chemistry / CRISPR-Associated Proteins - metabolism / CRISPR-Cas Systems - genetics / Enzymes / Humanities and Social Sciences / Leptotrichia - enzymology / letter / Methods / multidisciplinary / Mutation / Nucleic acids / Proteins / Ribonucleases - chemistry / Ribonucleases - metabolism / Ribonucleic acid / RNA / RNA Cleavage / RNA sequencing / RNA, Bacterial - genetics / RNA, Bacterial - metabolism / RNA, Guide, CRISPR-Cas Systems - genetics / RNA, Guide, CRISPR-Cas Systems - metabolism / Science