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Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers


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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Figure 1: Csm6 is allosterically activated by oligoA nucleotides.
Figure 2: The CARF domain mediates allosteric activation of Csm6.
Figure 3: The Cas10 complex activates Csm6 via a diffusible second messenger.
Figure 4: The Palm domain of Cas10 generates cyclic hexaadenylate in vitro.
Figure 5: In vivo activity of Csm6 is dependent on cyclic oligoA and Cas10.
Figure 6: Proposed model for the molecular mechanism of type III CRISPR–Cas systems.

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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

Authors and Affiliations



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.

Corresponding author

Correspondence to Martin Jinek.

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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.

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

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 AfCsx3 resembles the CARF domain of TtCsm6.

Left: structure of AfCsx3 (Protein Data Bank (PDB) accession number 3WZI) bound to a tetraribonucleotide. Centre: structure of the CARF domain dimer of TtCsm6 (PDB 5FSH). Right: superimposition of AfCsx3 (grey) and TtCsm6 (pink and blue). The structures were aligned using PDBeFold38.

Extended Data Figure 2 Activation of Csm6 ribonucleases by oligoadenylates.

a, TtCsm6 RNase activity assay in the presence of 3′-hydroxylated oligoadenylates ranging from A3 to A10. b, SDS–PAGE analysis of purified WT and mutant Csm6 proteins from T. thermophilus (TtCsm6), M. thermautotrophicus (MtCsm), and E. italicus (EiCsm6). c, d, MtCsm6 and EiCsm6 RNase activity assays in the presence of 3′-hydroxylated oligoadenylates ranging from A3 to A10. e, MtCsm6 RNase activity assay in the presence of oligoadenylates carrying 3′-hydroxyl (-OH) or 2′,3′-cyclic phosphate (>P) groups. f, TtCsm6 and EiCsm6 RNase activity assay in the presence of A4>P and A6>P. All data points represent the mean of three replicates ± s.e.m.

Extended Data Figure 3 Activation of EiCsm6 by A6>P.

a, EiCsm6 RNase assay in the presence of varying concentrations of A6>P. b, The log(dose)-versus-response curve and EC50 derived from assays in a. Errors are indicated as 95% confidence interval. All data points represent the mean of three replicates ± s.e.m.

Extended Data Figure 4 Conservation and structure of the CARF domain motif.

a, Multiple sequence alignment of the CARF domain motifs in Csm6. The polypeptide sequence of EiCsm6 (denoted with an arrow) was used for a BLAST search against the National Center for Biotechnology Information (NCBI) non-redundant protein sequences database and 19 best scoring hits with >95% query coverage were selected. The multiple sequence alignment was performed using Clustal Omega39 and visualized using ESPript 3 (ref. 40). Green asterisk denotes the conserved glutamine residue mutated to abrogate allosteric activation of EiCsm6. b, Zoom-in view of a structural model of the dimeric interface of the CARF domains in the EiCsm6 dimer. The model was generated using Phyre2 (ref. 41) on the basis of the TtCsm6 crystal structure (PDB 5FSH).

Extended Data Figure 5 Purification and crRNA-guided RNA cleavage by the EiCsm(1–5) complex.

a, E. italicus DSM15952 contains a CRISPR–Cas locus with nine cas genes followed by a repeat-spacer array. For heterologous expression of the EiCsm(1–5) complex, the genomic DNA fragment spanning genes csm1csm6 and cas6 and the first two repeat-spacer units in the CRISPR array was cloned into the expression plasmid. b, SDS–PAGE analysis of purified wild-type and mutant EiCsm(1–5) complexes. c, Denaturing gel of the products of a target RNase cleavage assay performed using a fluorophore-labelled target ssRNA substrate in the presence of WT or mutant EiCsm(1–5) complexes.

Extended Data Figure 6 The EiCsm(1–5) complex generates cyclic hexaadenylate.

a, Denaturing 20% polyacrylamide gel indicating time-resolved [α-32P]ATP oligomerization by the EiCsm(1–5) complex. b, Mass spectra of LC–MS peaks from the analysis of the products generated by the EiCsm(1–5)–dCsm3D32A and EiCsm(1–5)–dCas10Palm/dCsm3D32A complexes shown in Fig. 4c. The mass spectrum of synthetic A6>P standard is shown for comparison.

Extended Data Figure 7 LC–MS analysis of purified EiCsm(1–5) product.

a, c, e, LC–MS analysis of the HPLC-purified activator product generated by the EiCsm(1–5) complex as well as an A6>P standard and purified product spiked with the A6>P standard. b, d, f, Mass spectra of the peaks indicated in a, c, e.

Extended Data Figure 8 The EiCsm(1–5) complex generates cyclic hexaadenylate.

a, Left: LC–MS analysis of the nuclease S1 digested activator. Right: mass spectrum profile of the peak obtained after S1 digest. b, EiCsm6 RNase assay in the presence of varying concentrations of c-A6. c, Overlay of EC50 values obtained for A6>P and c-A6. Errors are indicated as 95% confidence interval and data points represent mean of three replicates ± s.e.m.

Extended Data Figure 9 The EiCsm(1–5) complex selectively incorporates adenosines into the activator.

a, Stimulation of EiCsm6 RNase activity by products generated by the EiCsm(1–5) complex in the presence of various ribonucleotide triphosphate combinations. b, LC–MS analysis of indicated samples from a. The elution profile (left) and mass spectrum (right) of the product remain unaltered upon addition of all four ribonucleotide triphosphates, indicating ATP selectivity of the EiCsm(1–5) complex.

Extended Data Figure 10 Proposed mechanism of cyclic oligoadenylate formation.

The Cas10 Palm domain initially catalyses 5′–3′ phosphodiester bond formation by a nucleophilic attack of the 3′-hydroxyl group of one ATP substrate molecule onto the α-phosphate group of a second ATP molecule, yielding a 5′-triphosphorylated dinucleotide and inorganic pyrophosphate (PPi). The dinucleotide is extended by addition of successive AMP moieties to the 3′ end to generate a linear oligonucleotide. Final cyclization involves the 3′-hydroxyl group attacking the α-phosphate group of the 5′-triphosphate moiety in the linear oligoadenylate intermediate. Notably, all reaction steps use the same catalytic mechanism and thus could be performed by a single catalytic active site in the Palm domain of Cas10.

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Niewoehner, O., Garcia-Doval, C., Rostøl, J. et al. Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers. Nature 548, 543–548 (2017).

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