A Thermostable Cas12b from Brevibacillus Leverages One-pot Detection of SARS-CoV-2 Variants of Concern
A Thermostable Cas12b from Brevibacillus Leverages One-pot Detection of SARS-CoV-2 Variants of Concern
ABSTRACT Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has the potential to transform diagnostics due to its programmability. However, many of the CRISPR-based detection methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. A complete one-pot detection reaction using alternative Cas effector endonucleases has been proposed to overcome these challenges. Yet, current approaches using Alicyclobacillus acidiphilus Cas12b (AapCas12b) are limited by its thermal instability at optimum reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction temperatures. Herein, we demonstrate that a novel Cas12b from Brevibacillus sp. SYP-B805 (referred to as BrCas12b) has robust trans-cleavage activity at ideal RT-LAMP conditions. A competitive profiling study of BrCas12b against Cas12b homologs from other bacteria genera underscores the potential of BrCas12b in the development of new diagnostics. As a proof-of-concept, we incorporated BrCas12b into an RT-LAMP-mediated one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs) to enable rapid, differential detection of SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) in 205 clinical samples. Notably, a BrCas12b detection signal was observed within 1-3 minutes of amplification, achieving an overall 98.1% specificity, 91.2% accuracy, and 88.1% sensitivity within 30 minutes. Significantly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, we combined the lyophilized one-pot reagents with a portable multiplexing device capable of interpreting fluorescence signals at a fraction of the cost of a qPCR system. With relaxed design requirements, one-pot detection, and simple instrumentation, this assay has the capability to advance future diagnostics.
Nguyen Long T.、Pizzano Brianna L.M.、Spacek Jan、Jain Piyush K.、Salemi Marco、Karasek Jan、Cash Melanie N.、Macaluso Nicolas C.、Dinglasan Rhoel R.
Department of Chemical Engineering, Herbert Wertheim College of Engineering, University of FloridaDepartment of Agricultural and Biological Engineering, College of Agricultural and Life Sciences, University of FloridaSparsek s.r.o.Department of Chemical Engineering, Herbert Wertheim College of Engineering, University of Florida||UF Health Cancer Center, University of FloridaDepartment of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida||Emerging Pathogens Institute, University of FloridaSCIERING s.r.o.Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida||Emerging Pathogens Institute, University of FloridaDepartment of Chemical Engineering, Herbert Wertheim College of Engineering, University of FloridaEmerging Pathogens Institute, University of Florida||Department of Infectious Diseases & Immunology, College of Veterinary Medicine, University of Florida
生物科学研究方法、生物科学研究技术微生物学分子生物学
Nguyen Long T.,Pizzano Brianna L.M.,Spacek Jan,Jain Piyush K.,Salemi Marco,Karasek Jan,Cash Melanie N.,Macaluso Nicolas C.,Dinglasan Rhoel R..A Thermostable Cas12b from Brevibacillus Leverages One-pot Detection of SARS-CoV-2 Variants of Concern[EB/OL].(2025-03-28)[2025-06-29].https://www.medrxiv.org/content/10.1101/2021.10.15.21265066.点此复制
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