BACKGROUND: Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. METHODS: Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs). FINDINGS: Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92·8% sensitivity, 99·4% specificity, and 96·7% accuracy within 10-30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99·1% and 95·1%, respectively. Interestingly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals. INTERPRETATION: This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19. FUNDING: This project was funded in part by the United States-India Science & Technology Endowment Fund- COVIDI/247/2020 (P.K.J.), Florida Breast Cancer Foundation- AGR00018466 (P.K.J.), National Institutes of Health- NIAID 1R21AI156321-01 (P.K.J.), Centers for Disease Control and Prevention- U01GH002338 (R.R.D., J.A.L., & P.K.J.), University of Florida, Herbert Wertheim College of Engineering (P.K.J.), University of Florida Vice President Office of Research and CTSI seed funds (M.S.), and University of Florida College of Veterinary Medicine and Emerging Pathogens Institute (R.R.D.).

Department of Agricultural and Biological Engineering College of Agricultural and Life Sciences University of Florida Gainesville FL USA

Department of Chemical Engineering Herbert Wertheim College of Engineering University of Florida Gainesville FL USA

Department of Chemical Engineering Herbert Wertheim College of Engineering University of Florida Gainesville FL USA; UF Health Cancer Center University of Florida Gainesville FL USA

Department of Pathology Immunology and Laboratory Medicine College of Medicine University of Florida Gainesville FL USA; Emerging Pathogens Institute University of Florida Gainesville FL USA

Emerging Pathogens Institute University of Florida Gainesville FL USA; Department of Environmental and Global Health College of Public Health and Health Professions University of Florida Gainesville FL USA

Emerging Pathogens Institute University of Florida Gainesville FL USA; Department of Infectious Diseases and Immunology College of Veterinary Medicine University of Florida Gainesville FL USA

SCIERING S R O Příkop 838 6 Zábrdovice Brno 62100 Czech Republic

Sparsek S R O Příkop 838 6 Zábrdovice Brno 60200 Czech Republic

medRxiv. 2021 Oct 18:2021.10.15.21265066. doi: 10.1101/2021.10.15.21265066. PubMed

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Joung J., Ladha A., Saito M., et al. Detection of SARS-CoV-2 with SHERLOCK one-pot testing. N Engl J Med. 2020;383(15):1492–1494. PubMed PMC

Nguyen P.Q., Soenksen L.R., Donghia N.M., et al. Wearable materials with embedded synthetic biology sensors for biomolecule detection. Nat Biotechnol. 2021 PubMed

de Puig H., Lee R.A., Najjar D., et al. Minimally instrumented SHERLOCK (miSHERLOCK) for CRISPR-based point-of-care diagnosis of SARS-CoV-2 and emerging variants. Sci Adv. 2021;7(32) PubMed PMC

Fozouni P., Son S., Diaz de Leon Derby M., et al. Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy. Cell. 2021;184(2):323–333. e9. PubMed PMC

Broughton J.P., Deng X., Yu G., et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020;38(7):870–874. PubMed PMC

Kaminski M.M., Abudayyeh O.O., Gootenberg J.S., Zhang F., Collins J.J. CRISPR-based diagnostics. Nat Biomed Eng. 2021;5(7):643–656. PubMed

Abudayyeh O.O., Gootenberg J.S. CRISPR diagnostics. Science. 2021;372(6545):914–915. PubMed

Chen J.S., Ma E., Harrington L.B., Da Costa M., et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018;360(6387):436–439. PubMed PMC

Li S.Y., Cheng Q.X., Wang J.M., et al. CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov. 2018;4:20. PubMed PMC

Li L., Li S., Wu N., et al. HOLMESv2: a CRISPR-Cas12b-assisted platform for nucleic acid detection and DNA methylation quantitation. ACS Synth Biol. 2019;8(10):2228–2237. PubMed

Gootenberg J.S., Abudayyeh O.O., Lee J.W., et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017;356(6336):438–442. PubMed PMC

Kellner M.J., Koob J.G., Gootenberg J.S., Abudayyeh O.O., Zhang F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc. 2019;14(10):2986–3012. PubMed PMC

Teng F., Cui T.T., Feng G.H., et al. Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov. 2018;4 PubMed PMC

Cofsky J.C., Karandur D., Huang C.J., Witte I.P., Kuriyan J., Doudna J.A. CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks. Elife. 2020;9 PubMed PMC

Notomi T., Okayama H., Masubuchi H., et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12):E63. PubMed PMC

Tian Y., Liu R.R., Xian W.D., Xiong M., Xiao M., Li W.J. A novel thermal Cas12b from a hot spring bacterium with high target mismatch tolerance and robust DNA cleavage efficiency. Int J Biol Macromol. 2020;147:376–384. PubMed

Li Z., Zhao W., Ma S., Li Z., Yao Y., Fei T. A chemical-enhanced system for CRISPR-Based nucleic acid detection. Biosens Bioelectron. 2021;192 PubMed PMC

Hardinge P., Murray J.A.H. Full dynamic range quantification using loop-mediated amplification (LAMP) by combining analysis of amplification timing and variance between replicates at low copy number. Sci Rep. 2020;10(1) Uk. PubMed PMC

Liu T.Y., Knott G.J., Smock D.C.J., et al. Accelerated RNA detection using tandem CRISPR nucleases. Nat Chem Biol. 2021;17(9):982. PubMed PMC

Ali Z., Aman R., Mahas A., et al. iSCAN: an RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2. Virus Res. 2020;288 PubMed PMC

Young R.M., Solis C.J., Barriga-Fehrman A., et al. Smartphone screen testing, a novel pre-diagnostic method to identify SARS-CoV-2 infectious individuals. Elife. 2021;10 PubMed PMC

Fontanet A., Autran B., Lina B., Kieny M.P., Karim S.S.A., Sridhar D. SARS-CoV-2 variants and ending the COVID-19 pandemic. Lancet. 2021;397(10278):952–954. PubMed PMC

Tregoning J.S., Flight K.E., Higham S.L., Wang Z., Pierce B.F. Progress of the COVID-19 vaccine effort: viruses, vaccines and variants versus efficacy, effectiveness and escape. Nat Rev Immunol. 2021;21(10):626–636. PubMed PMC

Chookajorn T., Kochakarn T., Wilasang C., Kotanan N., Modchang C. Southeast Asia is an emerging hotspot for COVID-19. Nat Med. 2021;27(9):1495–1496. PubMed

Bhoyar R.C., Jain A., Sehgal P., et al. High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next-generation sequencing. PLOS One. 2021;16(2) PubMed PMC

Otu A., Agogo E., Ebenso B. Africa needs more genome sequencing to tackle new variants of SARS-CoV-2. Nat Med. 2021;27(5):744–745. PubMed

Chiara M., D'Erchia A.M., Gissi C., et al. Next generation sequencing of SARS-CoV-2 genomes: challenges, applications and opportunities. Brief Bioinform. 2021;22(2):616–630. PubMed PMC