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. 2021 Mar 19;59(4):e02402-20.
doi: 10.1128/JCM.02402-20. Print 2021 Mar 19.

A Scalable, Easy-to-Deploy Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material

Jennifer N Rauch #  1   2 , Eric Valois #  1   2 , Sabrina C Solley  1 , Friederike Braig  1 , Ryan S Lach  1 , Morgane Audouard  1   2 , Jose Carlos Ponce-Rojas  1 , Michael S Costello  1 , Naomi J Baxter  1 , Kenneth S Kosik  1   2 , Carolina Arias  1   2 , Diego Acosta-Alvear #  3   2 , Maxwell Z Wilson #  3   2   4
Affiliations

A Scalable, Easy-to-Deploy Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material

Jennifer N Rauch et al. J Clin Microbiol. .

Abstract

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly trained personnel, and large upfront investment. Here, we showcase an orthogonal pipeline we call CREST (Cas13-based, rugged, equitable, scalable testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows a binary interpretation of results. CREST may provide a point-of-care solution to increase the distribution of COVID-19 surveillance.

Keywords: COVID-19; CRISPR; Cas13; SARS-CoV-2; point of care; testing.

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Figures

FIG 1
FIG 1
Overview of Cas13-based detection methods and CREST modifications. (i to iii) Standard sample collection, RNA extraction, and reverse transcription. (iv) Amplification using cost-effective Taq polymerase and portable thermocyclers instead of isothermal reactions. (v) Transcription and Cas13 activation are followed by fluorescence detection of dequenched poly(U) cleavage reporter visualized with a blue LED (∼495 nm) and orange filter or other fluorescence detection system.
FIG 2
FIG 2
Detection of SARS-CoV-2 RNA using CREST. (A) The mini-PCR mini16 thermocycler and P51 molecular fluorescence visualizer used in this study. Both are portable, can be operated with batteries, and have minimal footprint. (B) Fluorescence visualization of N1, N2, and N3 synthetic targets using the P51 visualizer.
FIG 3
FIG 3
Comparative analysis of method sensitivity and reagent cost per test. (A) (Left) Comparison of method sensitivity using a quantitative fluorescence detection instrument. (Right) RT-PCR + Cas13 detection visualized with lateral flow strips. (B) Associated costs of reagents per test of each testing method (excluding up-front instrumentation costs). A test is defined as a single sample run in triplicate.
FIG 4
FIG 4
Comparative analysis between CREST and TaqMan for SARS-CoV-2 N1 and N2 sites and RNase P. De-identified human samples are shown as gray circles. Error bars show standard deviations (n = 4). Dotted lines indicate the detection threshold for each assay.
FIG 5
FIG 5
Confusion matrix of combined results from Santa Barbara County Department of Public Health, UCSF, and UCSB asymptomatic clinical sample sets.
See this image and copyright information in PMC

References

    1. Mizumoto K, Kagaya K, Zarebski A, medRxiv GC. 2020. Estimating the asymptomatic ratio of 2019 novel coronavirus onboard the Princess Cruises ship, 2020. medrxiv.org.
    1. Hu Z, Song C, Xu C, Jin G, Chen Y, Xu X, Ma H, Chen W, Lin Y, Zheng Y, Wang J, Hu Z, Yi Y, Shen H. 2020. Clinical characteristics of 24 asymptomatic infections with COVID-19 screened among close contacts in Nanjing, China. Sci China Life Sci 63:706–711. doi:10.1007/s11427-020-1661-4. - DOI - PMC - PubMed
    1. Wölfel R, Corman VM, Guggemos W, Seilmaier M, Zange S, Müller MA, Niemeyer D, Jones TC, Vollmar P, Rothe C, Hoelscher M, Bleicker T, Brünink S, Schneider J, Ehmann R, Zwirglmaier K, Drosten C, Wendtner C. 2020. Virological assessment of hospitalized patients with COVID-2019. Nature 581:465–469. doi:10.1038/s41586-020-2196-x. - DOI - PubMed
    1. He X, Lau EHY, Wu P, Deng X, Wang J, Hao X, Lau YC, Wong JY, Guan Y, Tan X, Mo X, Chen Y, Liao B, Chen W, Hu F, Zhang Q, Zhong M, Wu Y, Zhao L, Zhang F, Cowling BJ, Li F, Leung GM. 2020. Temporal dynamics in viral shedding and transmissibility of COVID-19. Nat Med 26:672–675. doi:10.1038/s41591-020-0869-5. - DOI - PubMed
    1. Verity R, Okell LC, Dorigatti I, Winskill P, Whittaker C, Imai N, Cuomo-Dannenburg G, Thompson H, Walker PGT, Fu H, Dighe A, Griffin JT, Baguelin M, Bhatia S, Boonyasiri A, Cori A, Cucunubá Z, FitzJohn R, Gaythorpe K, Green W, Hamlet A, Hinsley W, Laydon D, Nedjati-Gilani G, Riley S, van Elsland S, Volz E, Wang H, Wang Y, Xi X, Donnelly CA, Ghani AC, Ferguson NM. 2020. Estimates of the severity of coronavirus disease 2019: a model-based analysis. Lancet Infect Dis 20:669–677. doi:10.1016/S1473-3099(20)30243-7. - DOI - PMC - PubMed