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

By Omar Abudayyeh, Jonathan Gootenberg, Silvana Konermann, J. Keith Joung, I. Slaymaker, David B. T. Cox, Sergey A. Shmakov, Kira S. Makarova, E. Semenova, L. Minakhin and 5 more. Science, 2016

2,074 citations recorded by the source corpus.

INTRODUCTION Almost all archaea and about half of bacteria possess clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated genes (Cas) adaptive immune systems, which protect microbes against viruses and other foreign DNA. All functionally characterized CRISPR systems have been reported to target DNA, with some multicomponent type III systems also targeting RNA. The putative class 2 type VI system, which has not been functionally characterized, encompasses the single-effector protein C2c2, which contains two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains commonly associated with ribonucleases (RNases), suggesting RNA-guided RNA-targeting function. RATIONALE Existing studies have only established a role for RNA interference, in addition to DNA interference, in the multicomponent type III-A and III-B systems. We investigated the possibility of C2c2-mediated RNA inference by heterologously expressing C2c2 locus from Leptotrichia shahii (LshC2c2) in the model system Escherichia coli. The ability of LshC2c2 to protect against MS2 single-stranded RNA (ssRNA) phage infection was assessed by using every possible spacer sequence against the phage genome. We next developed protocols to reconstitute purified recombinant LshC2c2 protein and test its biochemical activity when incubated with its mature CRISPR RNA (crRNA) and target ssRNA. We systematically evaluated the parameters necessary for cleavage. Last, to demonstrate the potential utility of the LshC2c2 complex for RNA targeting in living bacterial cells, we guided it to knockdown red fluorescent protein (RFP) mRNA in vivo. RESULTS This work demonstrates the RNA-guided RNase activity of the putative type VI CRISPR-effector LshC2c2. Heterologously expressed C2c2 can protect E. coli from MS2 phage, and by screening against the MS2 genome, we identified a H (non-G) protospacer flanking site (PFS) following the RNA target site, which was confirmed by targeting a complementary sequence in the β-lactamase transcript followed by a degenerate nucleotide sequence. Using purified LshC2c2 protein, we demonstrate that C2c2 and crRNA are sufficient in vitro to achieve RNA-guided, PFS-dependent RNA cleavage. This cleavage preferentially occurs at uracil residues in ssRNA regions and depends on conserved catalytic residues in the two HEPN domains. Mutation of these residues yields a catalytically inactive RNA-binding protein. The secondary structure of the crRNA direct repeat (DR) stem is required for LshC2c2 activity, and mutations in the 3′ region of the DR eliminate cleavage activity. Targeting is also sensitive to multiple or consecutive mismatches in the spacer:protospacer duplex. C2c2 targeting of RFP mRNA in vivo results in reduced fluorescence. The knockdown of the RFP mRNA by C2c2 slowed E. coli growth, and in agreement with this finding, in vitro cleavage of the target RNA results in “collateral,” nonspecific cleavage of other RNAs present in the reaction mix. CONCLUSION LshC2c2 is a RNA-guided RNase which requires the activity of its two HEPN domains, suggesting previously unidentified mechanisms of RNA targeting and degradation by CRISPR systems. Promiscuous RNase activity of C2c2 after activation by the target slows bacterial growth and suggests that C2c2 could protect bacteria from virus spread via programmed cell death and dormancy induction. A single-effector RNA targeting system has the potential to serve as a general chassis for molecular tools for visualizing, degrading, or binding RNA in a programmable, multiplexed fashion. C2c2 is an RNA-guided RNase that provides protection against RNA phage. CRISPR-C2c2 from L. shahii can be reconstituted in E. coli to mediate RNA-guided interference of the RNA phage MS2. Biochemical characterization of C2c2 reveals crRNA-guided RNA cleavage facilitated by the two HEPN nuclease domains. Binding of the target RNA by C2c2-crRNA also activates a nonspecific RNase activity, which may lead to promiscuous cleavage of RNAs without complementarity to the crRNA guide sequence. The clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated genes (Cas) adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the class 2 type VI CRISPR-Cas effector C2c2 and demonstrate its RNA-guided ribonuclease function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis shows that C2c2 is guided by a single CRISPR RNA and can be programmed to cleave single-stranded RNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains, mutations of which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/6a6fd5639b4e51e0e2a4af3a0bee616debcf4b5c

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Genetics

In Molecular Biology

Citations in the corpus

Transcriptome engineering with RNA-targeting Type VI-D CRISPR effectors (2018, positive): “Further, RNA target cleavage did not appear to depend on the protospacer flanking sequence (PFS) (Figure 3A) in contrast to other RNA-targeting class 2 systems, which require a 30-H (Abudayyeh et al., 2016) or a double-sided, DR-proximal 50-D and 30-NAN or NNA (Smargon et al.”
CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing (2019, methodology): “For instance, CRISPR–Cas has been used to introduce new sequences into the genome (1–3), activate (4,5) or repress (6) transcription, as an enrichment tool for sequencing (7,8), for targeted hypermutation (9), as a diagnostic tool (10), to perform whole-organism lineage tracing (11), to target RNA m”
CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA (2018, methodology): “12 Recently, this characteristic of Cas13a was successfully employed for rapid and sensitive nucleic acid detection.”
The Biology of CRISPR-Cas: Backward and Forward. (2018, positive): “Cas13 enzymes are programmed by crRNA and activated by target ssRNA but do not require tracrRNA (Abudayyeh et al., 2016; Liu et al., 2017b; Shmakov et al., 2015).”
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).”
Nucleic acid detection with CRISPR-Cas13a/C2c2 (2017, positive): “Upon recognition of its RNA target, activated Cas13a engages in “collateral” cleavage of nearby non-targeted RNAs (10).”
Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 (2018, positive): “The RNA-targeting CRISPR-associated enzyme Cas13(1, 2) has recently been adapted for such purpose.”
Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy (2020, neutral): “…could be explained by different quantification techniques of the target RNA or by considerable secondary structure in the viral RNA (Manfredonia et al., 2020; Sanders et al., 2020) that could reduce the accessibility of the target RNA for the RNP (Abudayyeh et al., 2017; Abudayyeh et al., 2016).”
New CRISPR-Cas systems from uncultivated microbes (2016, background): “In addition to Cas9, only three families of class 2 Cas effector proteins have been discovered and experimentally validated: Cpf1, C2c1, and C2c2 4,24,25 .”