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Accelerated RNA detection using tandem CRISPR nucleases

Nature Chemical Biology volume 17, pages 982–988 (2021)Cite this article

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A Publisher Correction to this article was published on 06 October 2021

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Abstract

Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR–Cas nucleases offer programmable RNA-guided RNA recognition that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper their detection sensitivity and speed. Here, we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 molecules per µl of RNA in 20 min. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA extracted from respiratory swab samples with quantitative reverse transcriptase PCR (qRT–PCR)-derived cycle threshold (Ct) values up to 33, using a compact detector. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables sensitive, direct RNA detection in a format that is amenable to point-of-care infection diagnosis as well as to a wide range of other diagnostic or research applications.

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Fig. 1: Activation and inactivation of TtCsm6 in an LbuCas13a–TtCsm6 assay.
Fig. 2: Effect of single 2′-activator modifications on Csm6-based signal generation in an RNA detection assay.
Fig. 3: Programmability and benchmarking of the LbuCas13a–TtCsm6 assay.
Fig. 4: Implementing FIND-IT on a compact fluorescence detector for analysis of COVID-19 samples.

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Data availability

The plasmid used to express MBP-tagged LbuCas13a (p2CT-His-MBP-Lbu_C2c2_WT) is available from Addgene (83482). Plasmids used for expression of SUMO-tagged LbuCas13a (pGJK_His-SUMO-LbuCas13a), His-tagged EiCsm6 (pET28a_His-TEV-EiCsm6) and His-SUMO-tagged TtCsm6 (pET_His6-SUMO-TEV-TtCsm6) will be available from Addgene (172488, 172487 and 172486). Sequences of RNA oligonucleotides are provided in Supplementary Table 3. Source data are provided with this paper.

Code availability

All custom data analysis for the limit of detection analysis for 20-replicate FIND-IT experiments and the code used for mathematical modeling of the Cas13–Csm6 reaction and illumination correction of images are available at https://github.com/jackdesmarais/FIND-IT. An archived version is available at https://doi.org/10.5281/zenodo.4921924 (ref. 40).

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Acknowledgements

This work was supported by DARPA under award number N66001-20-2-4033. The views, opinions and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the Department of Defense or the US Government. The work was also supported by the Howard Hughes Medical Institute (HHMI) and the National Institutes of Health (NIH) (R01GM131073 and DP5OD021369 to P.D.H., R01GM127463 to D.F.S., and 5R61AI140465-03 and R33AI140465 to J.A.D., D.A.F. and M.O.). A mass spectrometer was also purchased with NIH support (grant 1S10OD020062-01). This work was made possible by a generous gift from an anonymous private donor in support of the ANCeR diagnostics consortium. We also thank the David & Lucile Packard Foundation, the Shurl and Kay Curci Foundation and Fast Grants for their generous support of this project. We thank Integrated DNA Technologies and Synthego Corporation for support with oligonucleotide modifications and synthesis and Shanghai ChemPartner for expression and purification of the EiCsm6 protein. The computational aspects of this work related to SARS-CoV-2 guide RNA design were supported by the AWS Diagnostic Development Initiative via computational credit. We thank QB3 MacroLabs for subcloning TtCsm6, A.J. Aditham for assistance purifying TtCsm6 and D. Colognori for helpful discussions. J.A.D. is an HHMI investigator. G.J.K. acknowledges support from the NHMRC (Investigator Grant, EL1, 1175568). B.W.T. is supported by a National Science Foundation (NSF) Graduate Fellowship. P.F. was supported by the NIH/NIAID (F30AI143401). M.D.d.L.D. was supported by the UC MEXUS-CONACYT Doctoral Fellowship. K.S.P. and C.Z. were supported by Gladstone Institutes, the Chan Zuckerberg Biohub and a gift from Ed and Pam Taft. The pET_His6-SUMO-TEV LIC cloning vector (1S) used for subcloning the TtCsm6 expression vector was a gift from S. Gradia (Addgene, 29659). A gene fragment (gBlock) corresponding to nucleotides 27222-29890 of the SARS-CoV-2 Wuhan-Hu-1 variant genome (MN908947.2) was a gift from E. Connelly in the lab of C. Craik (University of California, San Francisco). The IGI Testing Consortium was supported by the David & Lucile Packard Foundation, the Shurl and Kay Curci Foundation, the Julia Burke Foundation and several anonymous donors. We thank all members of the IGI Testing Consortium at the University of California, Berkeley, for establishing the IGI testing lab and enabling use of deidentified clinical samples and data for this research study; a full list of the members, as of 6 July 2021, is provided in Supplementary Note 2.

Author information

Authors and Affiliations

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

    Tina Y. Liu, Gavin J. Knott, Dylan C. J. Smock, John J. Desmarais, Shrutee Jakhanwal, Emeric J. Charles, Brittney W. Thornton, Shineui Kim, Eli J. Dugan, David F. Savage & Jennifer A. Doudna

  2. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA

    Tina Y. Liu, Gavin J. Knott, Dylan C. J. Smock, Shrutee Jakhanwal, Noam Prywes, Shreeya Agrawal, Shineui Kim, Eli J. Dugan, Jennifer R. Hamilton, Enrique Lin-Shiao, Elizabeth C. Stahl, Connor A. Tsuchida, Erica A. Moehle, Petros Giannikopoulos, Matthew McElroy, Shana McDevitt, Arielle Zur, Iman Sylvain, Alison Ciling, Madeleine Zhu, Clara Williams, Alisha Baldwin, Liana F. Lareau, Patrick D. Hsu, David F. Savage & Jennifer A. Doudna

  3. Monash Biomedicine Discovery Institute, Department of Biochemistry & Molecular Biology, Monash University, Victoria, Australia

    Gavin J. Knott

  4. Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA

    Sungmin Son, Abdul Bhuiya, Shreeya Agrawal, María Díaz de León Derby, Andrew R. Harris, Daniel A. Fletcher, Liana F. Lareau & Patrick D. Hsu

  5. UC Berkeley, UC San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA, USA

    Abdul Bhuiya, María Díaz de León Derby & Daniel A. Fletcher

  6. Department of Physics and Astronomy, San José State University, San José, CA, USA

    Neil A. Switz

  7. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Maxim Armstrong & Jennifer A. Doudna

  8. Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, USA

    Parinaz Fozouni, Jeffrey Shu, Stephanie I. Stephens, G. Renuka Kumar, Chunyu Zhao, Katherine S. Pollard & Melanie Ott

  9. Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA

    Parinaz Fozouni

  10. Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA

    Parinaz Fozouni

  11. Department of Medicine, University of California, San Francisco, San Francisco, CA, USA

    Parinaz Fozouni, Jeffrey Shu, Stephanie I. Stephens, G. Renuka Kumar & Melanie Ott

  12. Chan-Zuckerberg Biohub, San Francisco, CA, USA

    Chunyu Zhao, Katherine S. Pollard & Daniel A. Fletcher

  13. Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA

    Amanda Mok & Patrick D. Hsu

  14. QB3/Chemistry Mass Spectrometry Facility, University of California, Berkeley, Berkeley, CA, USA

    Anthony T. Iavarone

  15. Wainamics Inc., Pleasanton, CA, USA

    Arturo M. Escajeda, Roger McIntosh & Ming X. Tan

  16. Department of Epidemiology & Biostatistics, University of California, San Francisco, San Francisco, CA, USA

    Katherine S. Pollard

  17. California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA

    Daniel A. Fletcher, Patrick D. Hsu & Jennifer A. Doudna

  18. Berkeley Stem Cell Center, University of California, Berkeley, Berkeley, CA, USA

    Patrick D. Hsu

  19. Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA

    Jennifer A. Doudna

  20. Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA

    Jennifer A. Doudna

  21. Gladstone Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA, USA

    Jennifer A. Doudna

Authors
  1. Tina Y. Liu
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  2. Gavin J. Knott
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  3. Dylan C. J. Smock
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  4. John J. Desmarais
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  5. Sungmin Son
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  6. Abdul Bhuiya
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  7. Shrutee Jakhanwal
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  10. María Díaz de León Derby
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  11. Neil A. Switz
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  12. Maxim Armstrong
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  13. Andrew R. Harris
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  14. Emeric J. Charles
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  15. Brittney W. Thornton
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  16. Parinaz Fozouni
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  17. Jeffrey Shu
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  18. Stephanie I. Stephens
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  19. G. Renuka Kumar
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  20. Chunyu Zhao
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  21. Amanda Mok
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  22. Anthony T. Iavarone
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  23. Arturo M. Escajeda
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  24. Roger McIntosh
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  25. Shineui Kim
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  26. Eli J. Dugan
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  27. Katherine S. Pollard
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  28. Ming X. Tan
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  29. Melanie Ott
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  30. Daniel A. Fletcher
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  31. Liana F. Lareau
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  32. Patrick D. Hsu
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  33. David F. Savage
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  34. Jennifer A. Doudna
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Consortia

IGI Testing Consortium

  • Jennifer R. Hamilton
  • , Enrique Lin-Shiao
  • , Elizabeth C. Stahl
  • , Connor A. Tsuchida
  • , Erica A. Moehle
  • , Petros Giannikopoulos
  • , Matthew McElroy
  • , Shana McDevitt
  • , Arielle Zur
  • , Iman Sylvain
  • , Alison Ciling
  • , Madeleine Zhu
  • , Clara Williams
  •  & Alisha Baldwin

Contributions

T.Y.L., J.A.D. and G.J.K. conceived the study. G.J.K, D.C.J.S. and T.Y.L. expressed and purified proteins. T.Y.L. and D.C.J.S. performed all biochemical experiments. T.Y.L., G.J.K., D.C.J.S., E.J.C., B.W.T., S.J., N.P. and S.A. performed initial experiments optimizing biochemical assays. T.Y.L. designed Csm6 activators with help from G.J.K. and N.P. G.J.K. prepared in vitro transcribed gBlock targets. J.J.D. analyzed the 20-replicate experiments and did modeling of the Cas13–Csm6 reaction. S.S., A.B., M.D.d.L.D., N.A.S., M.A., A.R.H., A.M.E. and R.M. developed the compact detector, data normalization method and software, with M.X.T. and D.A.F. providing supervision. T.Y.L., S.S. and J.J.D performed the statistical analysis for detection of clinical positives using FIND-IT. P.F., J.S., S.I.S., C.Z., A.M., G.J.K. and D.C.J.S. identified and analyzed crRNA sequences targeting SARS-CoV-2, with G.R.K., M.O., K.S.P. and L.F.L. providing supervision. A.T.I. performed MS data collection and analysis. S.K. and E.J.D. provided general experimental support. Authors from the IGI Testing Consortium (listed at the end of this paper) established, supervised and carried out qRT-PCR testing of clinical samples, interpreted the resulting Ct data, and provided deidentified clinical samples for FIND-IT testing. J.R.H. from the IGI Testing Consortium also performed experiments converting PCR-derived Ct values to copies of RNA per µl. T.Y.L. wrote the draft of the manuscript with assistance from G.J.K. and J.A.D. All authors edited and approved the manuscript. D.F.S., P.D.H. and J.A.D. obtained funding with significant help from N.P. Overall supervision of the project was provided by T.Y.L., G.J.K., P.D.H., D.F.S. and J.A.D.

Corresponding authors

Correspondence to Patrick D. Hsu, David F. Savage or Jennifer A. Doudna.

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Competing interests

D.F.S. is a cofounder of Scribe Therapeutics and a scientific advisory board member of Scribe Therapeutics and Mammoth Biosciences. P.D.H. is a cofounder of Spotlight Therapeutics and Moment Biosciences and serves on the board of directors and scientific advisory board and is a scientific advisory board member to Vial Health and Serotiny. The Regents of the University of California have patents issued and/or pending for CRISPR technologies on which J.A.D., T.Y.L., P.D.H., N.P., D.F.S., D.C.J.S., S.K., B.W.T., E.J.C., S.J. and G.J.K. are inventors. M.O., P.F., G.R.K., D.A.F., S.S. and N.A.S. have also filed patent applications related to this work. J.A.D. is a cofounder of Caribou Biosciences, Editas Medicine, Scribe Therapeutics, Intellia Therapeutics and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Mammoth Biosciences, Synthego, Algen Biotechnologies, Felix Biosciences and Inari. J.A.D. is a Director at Johnson & Johnson and has research projects sponsored by Biogen, Pfizer, AppleTree Partners and Roche.

Additional information

Peer review information Nature Chemical Biology thanks Catherine Freije and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Extended data

Extended Data Fig. 1 Direct activation of TtCsm6 by A4>P oligonucleotides, related to Fig. 1 and Fig. 2.

Direct activation of TtCsm6 with varying concentrations of the A4>P oligonucleotide (tetraadenylate with 2´,3´-cyclic phosphate). Mean fluorescence intensity with error bars indicating s.e.m. (n = 3) is shown in arbitrary fluorescence units (AU). The schematic above the graph is a representation of A4>P, with adenosines numbered A1-A4 from 5´ to 3´. Controls lacking TtCsm6 activator (0 µM A4>P) and containing only the fluorescent reporter (Reporter only) are shown for comparison. b) As in a, but with a single-fluoro A4>P oligonucleotide. The nucleotide bearing the 2´-F modification is colored pink in the schematic above the graph. c) As in a, but with a single deoxy A4>P oligonucleotide. The nucleotide bearing the 2´-H modification is colored white in the schematic above the graph. Controls shown in a are overlaid in b and c since all experiments in this figure were run in parallel.

Source data

Extended Data Fig. 2 Modeling of the Cas13-Csm6 detection reaction in the presence and absence of Csm6 activator degradation, related to Fig. 2.

a) A graph showing the modeled kinetics of fluorescent reporter cleavage by the HEPN domain of Csm6 when 0 copies per µl (cp/µl) to 6.25 × 105 cp/µl (1 pM) of target RNA are present and the CARF domain is active for cleavage of A4>P in a Cas13-Csm6 reaction (Csm6 CARF kcat = 0.05). A4>P is generated by Cas13-mediated cleavage of an A4-U6 oligonucleotide in the modeled reaction. b) As in a, but for a Cas13-Csm6 reaction in which the CARF domain of Csm6 is inactive for A4>P degradation (Csm6 CARF kcat = 0). In this condition, the HEPN domains of A4>P-bound Csm6 remain active for RNA cleavage over a longer time.

Extended Data Fig. 3 Triple-modified TtCsm6 activators lead to slow kinetics of fluorescent signal increase in a LbuCas13a-TtCsm6 detection assay.

a) LbuCas13a-TtCsm6 reaction with crRNA R004, 100 pM of a complementary target RNA (R010), and 4 µM of the triple-fluoro A4-U6 oligonucleotide. Controls without target RNA, TtCsm6 activator, and TtCsm6 are shown, as well as a reaction with only the fluorescent reporter in buffer (Reporter only). A schematic of the activator is shown above the graph, with 2´-F nucleotides colored pink. Mean normalized fluorescence values (F/F0) and s.e.m. (n = 3) are plotted as lines with error bars over the time course of the assay. b) As in a but with 4 µM of the triple-deoxy A4-U6 oligonucleotide. A schematic of the activator is shown above the graph with 2´-H nucleotides colored white. c) As in a but with 4 µM triple-O-methyl A4-U6 oligonucleotide. A schematic of the activator is shown above the graph with 2´-OMe nucleotides colored yellow. Controls in a that lack both the TtCsm6 activator and target RNA, or that contain only reporter are overlaid in b and c, as all experiments in this figure were run in parallel.

Source data

Extended Data Fig. 4 LbuCas13a-EiCsm6 detection assay with unmodified and single-fluoro activators at high target RNA concentrations, related to Fig. 2.

a) Detection assay using LbuCas13a complexed with two crRNAs, 604 and 612, and EiCsm6 with a A6-U5 oligonucleotide activator22. An in vitro transcribed RNA corresponding to a fragment of the SARS-CoV-2 genome (gblock) was added at concentrations ranging from 1.25 × 106 copies per µl (cp/µl) of RNA (2 pM) to 1.25 × 108 cp/µl) of RNA (200 pM). Control reactions that lack both target RNA and the EiCsm6 activator, or contain only the fluorescent reporter (Reporter only) are shown for comparison. Mean normalized fluorescence (F/F0) and s.e.m (n = 3) are plotted as lines with error bars over the reaction time course. A schematic of the A6-U5 activator is shown above the graph. b) As in a but using a single-fluoro A6-U5 oligonucleotide as an EiCsm6 activator. The adenosine bearing a 2´-F modification is colored pink in the schematic above the graph. Controls from a that lack both target RNA and EiCsm6 activator, or contain only the reporter are overlaid in the graph, as all experiments in this figure were run in parallel.

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Extended Data Fig. 5 Detection of SARS-CoV-2 RNA using LbuCas13a assembled with two or eight crRNA sequences, related to Fig. 3.

a) Detection of Twist synthetic SARS-CoV-2 RNA control using LbuCas13a loaded with two different crRNAs (604 and 612). The mean slope ± 95% CI of fluorescence signal increase over 120 min was plotted, with individual slopes shown as clear circles (n = 3). Pairwise comparisons of slopes to the control reaction with 0 copies per µl (cp/µl) of RNA was done by ANCOVA, as previously described21. Two-tailed P values are shown above each pairwise comparison. Non-significant P values (P > 0.05) are indicated with ‘ns’ above the comparison. Concentrations to the left of the dotted line were detected above the control. b) As in a, but with LbuCas13a complexed with eight crRNAs (604, 612, 542, 546, 564, 569, 588, 596) targeting the SARS-CoV-2 genome. c) Full 2-hour time course of the detection assays in a (left panel) and b (right panel), using LbuCas13a with two or eight crRNA sequences, respectively. The mean normalized fluorescence values (F/F0) ± s.e.m. are plotted as lines with error bars over the time course. The concentration of RNA present in each reaction is shown in the legend.

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Extended Data Fig. 6 Full time course data of LbuCas13a and LbuCas13a-TtCsm6 detection experiments in Fig. 3b,c.

a) Raw fluorescence signal from LbuCas13a-mediated cleavage of a fluorescent reporter in the presence of 0-125 copies/µl (cp/µl) of B.E.I. SARS-CoV-2 RNA. LbuCas13a is complexed with eight crRNAs targeting the SARS-CoV-2 genome (604, 612, 542, 546, 564, 569, 588, 596). The mean raw fluorescence values (arbitrary units, AU) ± s.e.m. are plotted as lines with error bars (n = 3). These data are used for detection analysis in Fig. 3b at 20 min and 118 min. b) Raw fluorescence signal from an LbuCas13a-TtCsm6 detection assay containing 0-125 cp/µl B.E.I. SARS-CoV-2 genomic RNA. The same crRNAs were used as in a. The mean raw fluorescence values (arbitrary units, AU) ± s.e.m. are plotted as lines with error bars (n = 3). c) Graph of the data in a, but with measurements normalized to the fluorescence at t = 6 min. The mean normalized fluorescence values (F/Ft=6) ± S.E.M. are shown as lines with error bars (n = 3). d) Graph of the data in b, but normalized to the fluorescence at t = 6 min. The mean normalized fluorescence values (F/Ft=6) ± s.e.m. are shown as lines with error bars (n = 3). These data were used in Fig. 3c for detection analysis at 20 min and 118 min. e) Same dataset as in c, but zoomed to the first 30 min of the LbuCas13a assay. f) Same dataset as in d, but zoomed to the first 30 min of the LbuCas13a-TtCsm6 assay.

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Extended Data Fig. 7 Full time course data corresponding to the 20-replicate LbuCas13a-TtCsm6 detection experiments in Fig. 3d.

a) 1-hour time course data for 20 replicates of FIND-IT reactions containing 125 copies per µl (cp/µl) of externally validated B.E.I. SARS-CoV-2 genomic RNA. Each set of 5 sample and 5 negative control (no target RNA) replicates were started simultaneously (R1-R5, R6-R10, R11-R15, R16-R20). Replicates with target RNA are plotted as black data points and lines, and control replicates lacking target RNA are plotted as pink data points and lines. Fluorescence was normalized to the fluorescence value at 6 min (F/Ft=6) to allow for temperature equilibration to 37 °C (See Extended Data Fig. 8). Each curve represents fluorescence data for a single replicate. b) As in a, but for sample reactions containing 63 cp/µl B.E.I. SARS-CoV-2 genomic RNA. c) As in a, but for sample reactions containing 31 cp/µl B.E.I. SARS-CoV-2 genomic RNA. d) As in a, but for sample reactions containing 16 cp/µl B.E.I. SARS-CoV-2 genomic RNA.

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Extended Data Fig. 8 Effect of normalizing fluorescence relative to different timepoints in a plate reader assay, related to Fig. 3.

Graphs of 10 replicates from the LbuCas13a-TtCsm6 assay in Fig. 3d with LbuCas13a bound to eight crRNA sequences (604, 612, 542, 546, 564, 569, 588, 596), single-fluoro A4-U6 oligonucleotide, and 125 copies per µl (cp/µl) of B.E.I. SARS-CoV-2 genomic RNA as the target. Black curves indicate replicates containing RNA, and pink curves indicate control replicates lacking RNA. Comparison of the normalization at 0, 4, and 6 min (t = 0, 4, 6) shows that normalizing a few minutes after the start of the plate reader assay to allow for temperature equilibration reduces the effect of initial signal fluctuations on data normalization.

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Extended Data Fig. 9 Testing clinical samples with FIND-IT assay in a compact fluorescence detector, related to Fig. 4.

a) Testing of SARS-CoV-2-positive clinical samples with FIND-IT using the compact detector in Fig. 4a–c. Images of the reaction wells were taken every 10 s for either 33 min (sample with a Ct value of 25) or 60 min (all other samples). Normalized fluorescence (F/F0) of a sample reaction (black lines) and a control reaction run in parallel (pink lines) are plotted in each graph. Ct values for the N gene, S gene, and ORF1ab locus were previously determined by qRT-PCR in the IGI testing laboratory, and the average Ct value is shown for each sample tested (individual Ct values are provided in Supplementary Table 5). A reaction was also prepared in which a sample with a Ct value of 33 was diluted 10-fold in water prior to analysis (‘Ct =33 (diluted 10-fold)’). b) As in a, but for clinical samples with Ct values > 37, which are considered negative for SARS-CoV-2, based on the qRT-PCR assay used by the IGI testing laboratory30. These data were used to determine the mean and s.d. of the fluorescence change of negative clinical samples (black) relative to their controls (pink; see Methods for details).

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Extended Data Fig. 10 Standard curve of qRT-PCR-derived Ct values versus sample RNA concentration.

a) Ct values obtained from qRT-PCR for a dilution series of the positive control RNA provided by the Thermo TaqPath combo kit. The highest concentration of sample added to the reaction is 1000 copies per µl (cp/µl) and lower RNA concentrations were obtained by diluting the concentrated sample two-fold down to ~2 cp/µl. Primers for the N gene of SARS-CoV-2 were used for amplification. Two replicates for each concentration of RNA are plotted as pink squares. b) As in a but with primers for ORF1ab locus of SARS-CoV-2. Replicates are plotted as green triangles. c) As in a but with primers for the S gene of SARS-CoV-2. Replicates are plotted as blue circles.

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Supplementary information

Supplementary Information

Supplementary Notes 1 and 2 and Tables 1–5.

Reporting Summary

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Source Data Fig. 1

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Photographs, unprocessed TIF images, normalized and raw data, statistical source data.

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Raw and normalized source data used for plots in Extended Data Fig. 7 and analysis in Fig. 3d.

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Illumination-corrected and normalized data, raw data.

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Liu, T.Y., Knott, G.J., Smock, D.C.J. et al. Accelerated RNA detection using tandem CRISPR nucleases. Nat Chem Biol 17, 982–988 (2021). https://doi.org/10.1038/s41589-021-00842-2

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