C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector

Abstract

The CRISPR-Cas adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the Class 2 type VI-A CRISPR-Cas effector C2c2 and demonstrate its RNA-guided RNase function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis show that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific † Correspondence should be addressed to zhang@broadinstitute.org (F.Z.) and koonin@ncbi.nlm.nih.gov (E.V.K.). * These authors contributed equally to this work. Almost all archaea and about half of bacteria possess Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes (CRISPR-Cas) adaptive immune systems (1, 2), which protect microbes from viruses and other invading DNA through three steps: (i) adaptation, i.e., insertion of foreign nucleic acid segments (spacers) into the CRISPR array in between pairs of direct repeats (DRs), (ii) transcription and processing of the CRISPR array to produce mature CRISPR RNAs (crRNAs), and (iii) interference, whereby Cas enzymes are guided by the crRNAs to target and cleave cognate sequences in the respective invader genomes (3-5). All CRISPR-Cas systems characterized to date follow these three steps, although the mechanistic implementation and proteins involved in these processes display extensive diversity. Supplementary Materials Supplementary Materials and MethodsThe CRISPR-Cas systems are broadly divided into two classes on the basis of the architecture of the interference module: Class 1 systems rely on multi-subunit protein complexes whereas Class 2 systems utilize single effector proteins (1). Within these two classes, types and subtypes are delineated according to the presence of distinct signature genes, protein sequence conservation, and organization of the respective genomic loci. (Fig. 1A). This resulted in a library of 3,473 spacer sequences (along with 490 non-targeting guides designed to have a Levenshtein distance of ≥8 with respect to the MS2 and E. coli genomes) which we inserted between pLshC2c2 direct repeats (DRs). After transformation in of this construct into E. coli, we infected cells with varying dilutions of MS2 (10 −1 , 10 −3 , and 10 −5 ) and analyzed surviving cells to determine the spacer sequences carried by cells that survived the infection. Cells carrying spacers that confer robust interference against MS2 are expected to proliferate faster than those that lack such sequences. Following growth for 16 hours, we identified a number of spacers that were consistently enriched across three independent infection replicas in both the 10 −1 and 10 −3 dilution conditions, suggesting that they enabled interference against MS2. Specifically, 147 and 150 spacers showed >1.25 log 2 -fold enrichment in all three replicates for the 10 −1 and 10 −3 phage dilutions, respectively; of these two groups of...

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“…Lastly, recent reports have described CRISPR-Cas systems with RNA-guided RNase activity. These include the type VI effectors Cas13a (C2c2) (Abudayyeh et al, 2016; East-Seletsky et al, 2016) and Cas13b (C2c6) (Smargon et al, 2017). RNA-targeted Cas proteins are beyond the scope of this review but hold tremendous promise for targeted manipulation of the transcriptome.…”

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