Letter

Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection

Received:
Accepted:
Published online:

Abstract

Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage1,2. Although most prokaryotic adaptive immune systems generally target DNA substrates3,4,5, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates6,7,8,9. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase)9,10. How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear. Here we show that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behaviour of the evolutionarily unrelated CRISPR enzyme Cpf1 (ref. 11). The two RNase activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow sensitive detection of cellular transcripts.

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Change history

  • Corrected online 12 October 2016

    The received date was updated.

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Acknowledgements

We thank the QB3 MacroLab for assistance with cloning of C2c2 constructs; N. Ma and K. Zhou for technical assistance; S. N. Floor, S. C. Strutt, A. V. Wright and M. L. Hochstrasser for critical reading of the manuscript; and members of the Doudna, Cate and Tjian laboratories for discussions. S.C.K. acknowledges support from the National Science Foundation Graduate Research Fellowship Program; M.R.O. is a recipient of a C. J. Martin Overseas Early Career Fellowship from the National Health and Medical Research Council of Australia. This work was supported in part by a Frontiers Science award from the Paul Allen Institute to J.A.D., the National Science Foundation (MCB-1244557 to J.A.D.), the California Institute for Regenerative Medicine (CIRM, RB4-06016 to R.T.), and the National Institutes of Health (P50-GM102706 to J.H.D.C). R.T. and J.A.D. are Investigators of the Howard Hughes Medical Institute. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine and Intellia Therapeutics and a scientific advisor to Caribou, Intellia, eFFECTOR Therapeutics, and Driver. A.E.S., M.R.O., S.C.K., J.H.D.C. and J.A.D. have filed a patent application related to this work.

Author information

Author notes

    • Alexandra East-Seletsky
    •  & Mitchell R. O’Connell

    These authors contributed equally to this work.

Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.

    • Alexandra East-Seletsky
    • , Mitchell R. O’Connell
    • , Jamie H. D. Cate
    • , Robert Tjian
    •  & Jennifer A. Doudna
  2. Department of Chemistry, University of California, Berkeley, California 94720, USA.

    • Spencer C. Knight
    • , Jamie H. D. Cate
    •  & Jennifer A. Doudna
  3. Department of Earth And Planetary Sciences, University of California, Berkeley, California 94720, USA.

    • David Burstein
  4. MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.

    • Jamie H. D. Cate
    •  & Jennifer A. Doudna
  5. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA.

    • Robert Tjian
  6. Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.

    • Robert Tjian
    •  & Jennifer A. Doudna
  7. Li Ka Shing Biomedical and Health Sciences Center, University of California, Berkeley, California 94720, USA.

    • Robert Tjian
  8. Innovative Genomics Initiative, University of California, Berkeley, California 94720, USA.

    • Jennifer A. Doudna

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Contributions

A.E.S., M.R.O. and S.C.K. conceived the study and designed experiments with input from J.H.D.C., R.T. and J.A.D. D.B. performed bioinformatic analyses. A.E.S. and M.R.O. executed all experimental work with assistance from S.C.K. All authors discussed the data and wrote the manuscript.

Competing interests

Authors A.E.S., M.R.O., S.C.K., J.H.D.C. and J.A.D. are inventors on a related patent application.

Corresponding author

Correspondence to Jennifer A. Doudna.

Reviewer Information

Nature thanks M. White, J. Wilusz and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

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    Supplementary Information

    This file contains Supplementary Figures 1-2, uncropped gel images.

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    Supplementary Data

    This file contains source data BAM files for sequencing reanalysis.

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