A Thermostable Cas12b from Brevibacillus Leverages One-Pot Discrimination of SARS-CoV-2 Variants of Concern

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. Here, for the first time, 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.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 characterized by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was 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) for discriminating SARS-CoV2 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 206 clinical samples. To facilitate dissemination and global implementation of the assay, a lyophilized 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. This technology enables realtime monitoring of RT-LAMP-mediated amplification and CRISPR-based reaction at a fraction of the cost of a qPCR system.Findings: Notably, a BrCas12b outperformed other common homologs in terms thermostability, displaying a melting temperature (Tm) of 63·4 o C, compared to AacCas12b (Tm= 55·3 o C) and AapCas12b (Tm= 58·3 o C). Consistent to this, a significantly higher trans-cleavage activity was observed at the temperature range of 60-65 o C that is desirable for RT-LAMP amplification. The BrCas12b detection signal was observed within 1-3 minutes of RTLAMP amplification, and the CRISPR-SPADE achieved 92·7% sensitivity, 99·4% specificity, and 96·7% accuracy within 10-30 minutes 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 (table 1). Interestingly, for samples with high viral load (C t value ≤ 30), 100% accuracy and sensitivity were attained (table S1).Interpretation: A competitive profiling study of BrCas12b against Cas12b homologs from other bacteria genera underscores the potential of BrCas12b in the development of new diagnostics. Current one-pot CRISPR-based approaches using AapCas12b are limited by its thermal instability at optimum RT-LAMP reaction temperatures. BrCas12b displays a robust transcleavage activity at ideal RT-LAMP conditions, perfect for designing a one-pot reaction system and for discriminating SARS-CoV-2 VOCs clinically. With relaxed design requirements, one-pot detection, lyophilized reagents, simple instrumentation, and our preliminary results indicate that CRISPR-SPADE has the capability to discriminate newer VOCs, including Omicron (B.1.1.529), and advance future diagnostics beyond COVID-19.Funding Information: This work was funded in part by the UF Herbert Wertheim College of Engineering (PKJ), the Preeminence Program of the University of Florida College of Veterinary Medicine (RRD), United States-India Science & Technology Endowment Fund (USISTEF/COVID I/247/2020; PKJ), Florida Breast Cancer Foundation (AGR00018466; PKJ), National Institutes of Health (NIAID 1R21AI156321-01; PKJ), and Centers for Disease Control and Prevention (U01GH002338; PKJ, RRD) Declaration of Interests: L.T.N. and P.K.J. are listed as inventors on the multiple patent applications related to the content of this work. J.S. and J.K. are both co-founders of Sparsek s.r.o. J.K. is also the founder of SCIERING s.r.o. R.R.D. and P.K.J are co-founders of Genable Biosciences, LLC. The remaining authors declare no competing interests.Ethics Approval Statement: The sample collection followed the guidelines approved by UF Institutional Review Board (IRB202000781) and through Evaluating the Molecular Epidemiology of Coronavirus (COVID-19) in Florida (IRB202000633).