Letter | Published:

Programmable RNA recognition and cleavage by CRISPR/Cas9

Nature volume 516, pages 263266 (11 December 2014) | Download Citation

Abstract

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA–DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage1,2,3,4,5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA4,5,6,7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

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Acknowledgements

We thank B. Staahl and K. Zhou for technical assistance, A. Iavarone for assistance with mass spectrometry measurements, Integrated DNA Technologies for the synthesis of DNA and RNA oligonucleotides, and members of the Doudna laboratory and J. Cate for discussions and critical reading of the manuscript. S.H.S. acknowledges support from the National Science Foundation and National Defense Science & Engineering Graduate Research Fellowship programs. A.E.-S. and B.L.O. acknowledge support from NIH NRSA trainee grants. Funding was provided by the NIH-funded Center for RNA Systems Biology (P50GM102706-03). J.A.D. is an Investigator of the Howard Hughes Medical Institute.

Author information

Author notes

    • Matias Kaplan

    Present address: Department of Agricultural and Biological Engineering, University of Florida, Gainesville, Florida 32611, USA.

Affiliations

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

    • Mitchell R. O’Connell
    • , Benjamin L. Oakes
    • , Alexandra East-Seletsky
    •  & Jennifer A. Doudna
  2. Department of Chemistry, University of California, Berkeley, California 94720, USA

    • Samuel H. Sternberg
    •  & Jennifer A. Doudna
  3. Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA

    • Matias Kaplan
    •  & Jennifer A. Doudna
  4. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • Jennifer A. Doudna

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Contributions

M.R.O. and S.H.S. conceived the project. M.R.O., B.L.O., S.H.S., A.E.-S. and M.K. conducted experiments. All authors discussed the data, and M.R.O., S.H.S., B.L.O. and J.A.D. wrote the manuscript.

Competing interests

J.A.D., M.R.O., B.L.O. and S.H.S. are inventors on a related patent application.

Corresponding author

Correspondence to Jennifer A. Doudna.

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https://doi.org/10.1038/nature13769

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