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

Abudayyeh, Omar O., Gootenberg, Jonathan S., Konermann, Silvana, Joung, Julia, Slaymaker, Ian M., Cox, David B. T., Shmakov, Sergey, Makarova, Kira S., Semenova, Ekaterina, Minakhin, Leonid, Severinov, Konstantin, Regev, Aviv, Lander, Eric S., Koonin, Eugene V., Zhang, Feng·2016·Science
DOI 10.1126/science.aaf5573PMC PMC5127784

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Abstract

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.

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  • A species-dependent protospacer flanking site (PFS), analogous to PAMs in DNA targets, is relevant for activation of Cas13 proteins.

    “A species-dependent protospacer flanking site (PFS), analogous to PAMs in DNA targets, is also of relevance for activation of Cas13 proteins [58].”

    From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [58]

    In “Reconstitution of the L. shahii C2c2 locus” (page unavailable), the paper reports that a 3′ PFS affects C2c2 targeting efficacy; its RNA-cleavage experiments also find a preference for A, U, or C over G. The evidence is for L. shahii C2c2, not species-dependent PFS requirements across Cas13 proteins. The paper also distinguishes PFS from PAM terminology rather than treating them as equivalent. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • CRISPR systems that work with RNA can be used to deactivate specific transcripts inside the cell selectively.

    “and, therefore, can be used to deactivate specific transcripts inside the cell in a selective way [14, 15].”

    From CRISPR–Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing (2022) · cites this paper as [14]

    In “C2c2 can be reprogrammed to mediate specific mRNA knockdown in vivo,” the paper reports crRNA-directed RFP mRNA knockdown in E. coli. However, “C2c2 cleaves collateral RNA in addition to crRNA-targeted ssRNA” reports nonspecific cleavage of other RNA, so the claim overstates how selective the deactivation is. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Cas13 is unique relative to other class 2 systems due to its ability to cleave ssRNA molecules homologous to crRNA, which is complemented with non-specific cleaving of other ssRNAs, similar to the Csm6 enzyme of type III systems.

    “Cas13 is unique relative to other class 2 systems due to its ability to cleave ssRNA molecules homologous to crRNA, which is complemented with non-specific cleaving of other ssRNAs, similar to the Csm6 enzyme of type III systems [57,58].”

    From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [58]

  • Cas13a is a class 2 CRISPR system.

    “Although type VI Cas13a is also a class 2 CRISPR system”

    From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [58]

  • Cas13a cleaves exclusively RNA through two HEPN domains, unlike Cas9 and Cas12a which cleave DNA.

    “it has the ability to cleave exclusively RNA through the activity of two HEPN domains [58], in contrast with the DNA cleaving ability of Cas9 and Cas12a.”

    From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [58]

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Metadata

Authors
Abudayyeh, O. O., Gootenberg, J. S., Konermann, S., Joung, J., Slaymaker, I. M., Cox, D. B. T., Shmakov, S., Makarova, K. S., Semenova, E., Minakhin, L., Severinov, K., Regev, A., Lander, E. S., Koonin, E. V., & Zhang, F.
Affiliations
  • Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
  • Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • Skolkovo Institute of Science and Technology, Skolkovo, 143025, Russia.
  • National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
  • Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
Venue
Science
Publisher
American Association for the Advancement of Science (AAAS)
Published
2016-08-05
Volume
353
Issue
6299
ISSN
  • 0036-8075
  • 1095-9203
DOI
10.1126/science.aaf5573
PMC
PMC5127784
Cited by
2,336
References
5
Language
en
Funders
  • National Institute of General Medical Sciences (T32GM007753)
  • U.S. Department of Health and Human Services
  • NIH (GM10407)
  • Russian Science Foundation (14-14-00988)
  • Skoltech
  • New York Stem Cell Foundation
  • National Institute of Mental Health (5DP1-MH100706, 1R01-MH110049)
  • NSF
  • New York Stem Cell
  • Simons
  • Paul G. Allen Family
  • Vallee Foundation

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