Abstract

RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference1,2,3 can efficiently knockdown RNAs, but it is prone to off-target effects4, and visualizing RNAs typically relies on the introduction of exogenous tags5. Here we demonstrate that the class 2 type VI6,7 RNA-guided RNA-targeting CRISPR–Cas effector Cas13a8 (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR–Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.

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Acknowledgements

We thank M. Alimova, D. Feldman, F. Chen, J. G. Doench, J. M. Engreitz, N. Habib, D. Tenen, A. Allen, R. Macrae, and R. Belliveau for discussions and support. O.A.A. is supported by a Paul and Daisy Soros Fellowship and a National Defense Science and Engineering Fellowship. J.S.G. is supported by a D.O.E. Computational Science Graduate Fellowship. A.R. is supported by the Howard Hughes Medical Institute. F.Z. is a New York Stem Cell Foundation-Robertson Investigator. F.Z. is supported by the National Institutes of Health through the National Institute of Mental Health (5DP1-MH100706 and 1R01-MH110049), the Howard Hughes Medical Institute, the New York Stem Cell, Simons, Paul G. Allen Family, and Vallee Foundations; and James and Patricia Poitras, Robert Metcalfe, and David Cheng.

Author information

Author notes

    • Omar O. Abudayyeh
    •  & Jonathan S. Gootenberg

    These authors contributed equally to this work.

Affiliations

  1. Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA

    • Omar O. Abudayyeh
    • , Jonathan S. Gootenberg
    • , Patrick Essletzbichler
    • , Julia Joung
    • , Vanessa Verdine
    • , David B. T. Cox
    • , Max J. Kellner
    • , Aviv Regev
    • , Eric S. Lander
    •  & Feng Zhang
  2. McGovern Institute for Brain Research at MIT, Cambridge, Massachusetts 02139, USA

    • Omar O. Abudayyeh
    • , Jonathan S. Gootenberg
    • , Patrick Essletzbichler
    • , Julia Joung
    • , Vanessa Verdine
    • , David B. T. Cox
    •  & Feng Zhang
  3. Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Omar O. Abudayyeh
    • , Jonathan S. Gootenberg
    • , Patrick Essletzbichler
    • , Julia Joung
    • , Vanessa Verdine
    • , David B. T. Cox
    •  & Feng Zhang
  4. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Omar O. Abudayyeh
    • , Jonathan S. Gootenberg
    • , Patrick Essletzbichler
    • , Julia Joung
    • , Vanessa Verdine
    • , David B. T. Cox
    •  & Feng Zhang
  5. Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Omar O. Abudayyeh
  6. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Jonathan S. Gootenberg
    •  & Eric S. Lander
  7. Departments of Genetics, Biology, and Chemistry, Stanford University, Stanford, California 94305, USA

    • Shuo Han
    •  & Alice Y. Ting
  8. Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota 55455, USA

    • Joseph J. Belanto
    •  & Daniel F. Voytas
  9. Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455, USA

    • Joseph J. Belanto
    •  & Daniel F. Voytas
  10. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • David B. T. Cox
    • , Aviv Regev
    •  & Eric S. Lander

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Contributions

O.O.A., J.S.G., and F.Z. conceived and designed the study. O.O.A. and J.S.G. participated in the design and execution of all experiments. P.E. performed biochemical characterization studies on the LwaCas13a protein. J.J. and V.V. prepared the vectors for arrayed knockdown screening experiments. J.J. also performed RNA immunoprecipitation experiments. D.B.T.C. assisted with cloning of constructs. M.J.K. performed the RNA integrity analysis. O.O.A. and J.S.G. analysed data. S.H. performed select microscopy experiments. J.J.B. performed the plant protoplast knockdown experiments. O.O.A., J.S.G., E.S.L., and F.Z. wrote the paper with input from D.F.V., A.Y.T., and A.R. and help from all authors.

Competing interests

Patent applications have been filed relating to work in this manuscript. F.Z. is an adviser for Editas Medicine and Horizon Discovery.

Corresponding author

Correspondence to Feng Zhang.

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

Extended data

Supplementary information

PDF files

  1. 1.

    Reporting Summary

  2. 2.

    Supplementary Information

    This file contains a discussion, notes 1-7 and references.

  3. 3.

    Supplementary Figure 1

    This file contains extended data figures 2a, b, c, e, g, h, and i.

  4. 4.

    Supplementary Table 1

    This file contains the guides used for in vivo experiments in this study.

  5. 5.

    Supplementary Table 2

    This file contains the plasmids used in this study.

  6. 6.

    Supplementary Table 3

    This file contains the shRNA used in this study.

  7. 7.

    Supplementary Table 4

    This file contains the ssRNA targets used in this study.

  8. 8.

    Supplementary Table 5

    This file contains the guides used for in vivo experiments in this study.

  9. 9.

    Supplementary Table 6

    This file contains the ssRNA targets and crRNAs used for the SHERLOCK experiments.

  10. 10.

    Supplementary Table 8

    This file contains the Commercial TaqMan probes used in this study.

  11. 11.

    Supplementary Table 9

    This file contains the Custom TaqMan probes used in this study.

  12. 12.

    Supplementary Table 10

    This file contains the Cas13a orthologs used in this study.

Excel files

  1. 1.

    Supplementary Table 7

    This file contains the guide sequences used in arrayed RNA knockdown screens.

About this article

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DOI

https://doi.org/10.1038/nature24049

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