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

Author:

Abudayyeh Omar O.1234,Gootenberg Jonathan S.2345,Konermann Silvana234,Joung Julia234,Slaymaker Ian M.234,Cox David B. T.12346,Shmakov Sergey78,Makarova Kira S.8,Semenova Ekaterina9,Minakhin Leonid9,Severinov Konstantin7910,Regev Aviv26,Lander Eric S.256,Koonin Eugene V.8,Zhang Feng1234

Affiliation:

1. Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

2. Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

3. McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA.

4. Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

5. Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.

6. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

7. Skolkovo Institute of Science and Technology, Skolkovo, 143025, Russia.

8. National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.

9. Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.

10. Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182, Russia.

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.

Funder

National Institute of General Medical Sciences

U.S. Department of Health and Human Services

NIH

Russian Science Foundation

Skoltech

New York Stem Cell Foundation

National Institute of Mental Health

NSF

New York Stem Cell

Simons

Paul G. Allen Family

Vallee Foundation

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

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