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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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).
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31 January 2020
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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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M.J.K., J.G.K., J.S.G., O.O.A., and F.Z. designed and conducted all experiments and wrote the manuscript.
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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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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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DOI: https://doi.org/10.1038/s41596-019-0210-2
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