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SHERLOCK: nucleic acid detection with CRISPR nucleases

Nature Protocols volume 14, pages 2986–3012 (2019)Cite this article

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An Author Correction to this article was published on 31 January 2020

This article has been updated

Abstract

Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR–Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.

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Fig. 1: Cas13 complex and collateral activity.
Fig. 2: Complete SHERLOCK experimental workflow.
Fig. 3: Considerations for primer and crRNA design.
Fig. 4: Protein purification workflow and expected results.
Fig. 5: Anticipated fluorescence and lateral flow results.

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Data and materials availability

Reagents from the Zhang lab are widely available to the academic community through Addgene, and additional information and resources can be found on the Zhang lab website (https://zlab.bio/) and GitHub (github.com/fengzhanglab).

Change history

  • 31 January 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank W. Blake and C. Brown for helpful feedback, and R. Macrae, R. Belliveau, E. Blackwell, and the entire Zhang lab for discussions and support. O.O.A. was supported by an NIH grant (F30 NRSA 1F30-CA210382). F.Z. was a New York Stem Cell Foundation–Robertson Investigator. F.Z. was supported by NIH grants (1R01-HG009761, 1R01-MH110049, and 1DP1-HL141201); the Howard Hughes Medical Institute; the New York Stem Cell and G. Harold and Leila Mathers foundations; the Poitras Center for Affective Disorders Research at MIT; the Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT; and J. and P. Poitras, R. Metcalfe, and D. Cheng.

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Authors and Affiliations

  1. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Max J. Kellner, Jeremy G. Koob, Jonathan S. Gootenberg, Omar O. Abudayyeh & Feng Zhang

  2. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    Jonathan S. Gootenberg, Omar O. Abudayyeh & Feng Zhang

  3. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA

    Jonathan S. Gootenberg, Omar O. Abudayyeh & Feng Zhang

  4. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Feng Zhang

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  1. Max J. Kellner
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  2. Jeremy G. Koob
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  4. Omar O. Abudayyeh
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  5. Feng Zhang
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Contributions

M.J.K., J.G.K., J.S.G., O.O.A., and F.Z. designed and conducted all experiments and wrote the manuscript.

Corresponding authors

Correspondence to Jonathan S. Gootenberg, Omar O. Abudayyeh or Feng Zhang.

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Competing interests

M.J.K., J.S.G., O.O.A., and F.Z. are co-inventors on patent applications filed by the Broad Institute relating to work in this article. J.S.G., O.O.A., and F.Z. are co-founders of Sherlock Biosciences. F.Z. is a co-founder and advisor of Beam Therapeutics, Editas Medicine, Pairwise Plants, and Arbor Biotechnologies. O.O.A. and J.S.G. are advisers for Beam Therapeutics. J.S.G. is a campus advisor for Benchling, Inc.

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Peer review information Nature Protocols thanks Tetsushi Sakum, Jin Wang and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Gootenberg, J. S. et al. Science 356, 438−442 (2017): https://doi.org/10.1126/science.aam9321

Gootenberg, J. S. et al. Science 360, 439−444 (2018): https://doi.org/10.1126/science.aaq0179

Myhrvold, C. et al. Science 360, 444−448 (2018): https://doi.org/10.1126/science.aas8836

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Kellner, M.J., Koob, J.G., Gootenberg, J.S. et al. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc 14, 2986–3012 (2019). https://doi.org/10.1038/s41596-019-0210-2

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