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

34 Pages Posted: 20 Jan 2022

See all articles by Long T. Nguyen

Long T. Nguyen

University of Florida - Department of Chemical Engineering

Nicolas C. Macaluso

University of Florida - Department of Chemical Engineering

Brianna L.M. Pizzano

University of Florida - Department of Chemical Engineering; University of Florida - Department of Agricultural and Biological Engineering

Melanie N. Cash

University of Florida - Department of Pathology, Immunology and Laboratory Medicine

Jan Spacek

Sparsek s.r.o. Příkop 838/6, Zábrdovice, 602 00

Jan Karasek

SCIERING s.r.o., Příkop 838/6, 621 00

Rhoel R. Dinglasan

University of Florida - Emerging Pathogens Institute

Marco Salemi

University of Florida - Department of Pathology, Immunology and Laboratory Medicine

Piyush K. Jain

University of Florida - Department of Chemical Engineering

More...

Abstract

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).

Keywords: CRISPR, CRISPR/Cas, CRISPR/Cas12b, COVID-19, SARS-COV-2, diagnostics, infectious diseases, variants of concern, detection, point-of-care, RT-LAMP, fluorescence

Suggested Citation

Nguyen, Long T. and Macaluso, Nicolas C. and Pizzano, Brianna L.M. and Cash, Melanie N. and Spacek, Jan and Karasek, Jan and Dinglasan, Rhoel R. and Salemi, Marco and Jain, Piyush K., A Thermostable Cas12b from Brevibacillus Leverages One-Pot Discrimination of SARS-CoV-2 Variants of Concern. Available at SSRN: https://ssrn.com/abstract=4008146 or http://dx.doi.org/10.2139/ssrn.4008146

Long T. Nguyen

University of Florida - Department of Chemical Engineering ( email )

Gainesville, FL
United States

Nicolas C. Macaluso

University of Florida - Department of Chemical Engineering ( email )

Gainesville, FL
United States

Brianna L.M. Pizzano

University of Florida - Department of Chemical Engineering ( email )

University of Florida - Department of Agricultural and Biological Engineering ( email )

Gainesville, FL
United States

Melanie N. Cash

University of Florida - Department of Pathology, Immunology and Laboratory Medicine ( email )

1600 SW Archer Rd
M509
Gainesville, FL 32610
United States

Jan Spacek

Sparsek s.r.o. Příkop 838/6, Zábrdovice, 602 00 ( email )

Brno
Czech Republic

Jan Karasek

SCIERING s.r.o., Příkop 838/6, 621 00 ( email )

Brno
Czech Republic

Rhoel R. Dinglasan

University of Florida - Emerging Pathogens Institute ( email )

Gainesville, FL
United States

Marco Salemi

University of Florida - Department of Pathology, Immunology and Laboratory Medicine ( email )

1600 SW Archer Rd
M509
Gainesville, FL 32610
United States

Piyush K. Jain (Contact Author)

University of Florida - Department of Chemical Engineering ( email )

Gainesville, FL
United States

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