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Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers

Nature volume 548, pages 543548 (31 August 2017) | Download Citation


In many prokaryotes, type III clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated (Cas) systems detect and degrade invasive genetic elements by an RNA-guided, RNA-targeting multisubunit interference complex. The CRISPR-associated protein Csm6 additionally contributes to interference by functioning as a standalone RNase that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 proteins are activated through a second messenger generated by the type III interference complex. Upon target RNA binding by the interference complex, its Cas10 subunit converts ATP into a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CRISPR-associated Rossmann fold (CARF) domain. CARF domain mutations that abolish allosteric activation inhibit Csm6 activity in vivo, and mutations in the Cas10 Palm domain phenocopy loss of Csm6. Together, these results point to an unprecedented mechanism for regulation of CRISPR interference that bears striking conceptual similarity to oligoadenylate signalling in mammalian innate immunity.

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We thank members of the Jinek and Marraffini laboratories for discussions and comments on the manuscript. We thank D. Swarts and P. Sledz for technical assistance and sharing reagents. We thank U. Manzau for technical assistance during A4>P synthesis and purification. We thank the Service for Mass Spectrometry of ETH Zurich for support with MS analysis. This study was supported by a Swiss National Science Foundation project grant to M.J. (SNSF 31003A_149393) and by funding from the Swiss National Competence Center for Research (NCCR) ‘RNA & Disease’ (to M.J. and J.H.). M.J. is an International Research Scholar of the Howard Hughes Medical Institute and Vallee Scholar of the Bert L & N Kuggie Vallee Foundation. C.G.-D. was supported by a Long-Term Fellowship from the European Molecular Biology Organization. J.T.R. was supported by a Boehringer Ingelheim Fonds PhD fellowship. L.A.M. is supported by the Rita Allen Scholars Program, a Burroughs Wellcome Fund PATH award, a National Institutes of Health Director’s New Innovator Award (1DP2AI104556-01), and a Howard Hughes Medical Institute-Simons Faculty Scholar Award.

Author information

Author notes

    • Ole Niewoehner
    •  & Carmela Garcia-Doval

    These authors contributed equally to this work.


  1. Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

    • Ole Niewoehner
    • , Carmela Garcia-Doval
    •  & Martin Jinek
  2. Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065-6399, USA

    • Jakob T. Rostøl
    •  & Luciano A. Marraffini
  3. Department of Chemistry and Applied Biosciences, Institute for Pharmaceutical Sciences, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland

    • Christian Berk
    •  & Jonathan Hall
  4. BIOLOG Life Science Institute GmbH, Flughafendamm 9a, D-28199 Bremen, Germany

    • Frank Schwede
  5. Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

    • Laurent Bigler


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O.N., C.G.-D., and M.J. conceived the study. O.N., C.G.-D., J.T.R., L.M., and M.J. designed experiments. O.N. expressed and purified recombinant Csm6 proteins, performed oligoA activation and ATPase assays, and performed enzymatic probing of the cyclic oligoA product. C.G.-D. expressed and purified recombinant EiCsm(1–5) complexes, performed oligoA activation assays, and assisted with LC–MS analysis. J.T.R. performed phage infection assays under supervision of L.M. C.B. synthesized 2′,3′-cyclic phosphate-terminated nucleotides and performed LC–MS analysis under supervision of J.H. F.S. synthesized 2′,3′-cyclic phosphate-terminated A4 nucleotide and advised on nucleotide chemistry. L.B. performed additional LC–MS analyses of Csm6 activators. O.N., C.G.-D., and M.J. wrote the manuscript, with input from the remaining authors.

Competing interests

F.S. is an employee of BIOLOG Life Science Institute GmbH, which markets synthetic nucleotides. The other authors declare no competing financial interests.

Corresponding author

Correspondence to Martin Jinek.

Reviewer Information Nature thanks S. Bailey, P. Kranzusch and R. Staals for their contribution to the peer review of this work.

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