Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids.

CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C-65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses, we introduce mutations to wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing thermostability. The one-pot detection assay utilizing the engineered BrCas12b, called SPLENDID (single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting. We validate SPLENDID clinically in 80 serum samples for hepatitis C virus (HCV) and 66 saliva samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and accuracy. We obtain results in as little as 20 min, and with the extraction process, the entire assay can be performed within an hour.

An extraction-free one-step CRISPR-assisted detection platform and a potential Streptococcus pneumoniae at-home self-testing kit.

A rapid, accurate, easy-to-use nucleic acid detection technology is essential for disease diagnosis and control. Herein, we improved CRISPR-top (cluster regularly interspaced short palindromic repeats-mediated testing in one-pot) to develop Extraction-free one-step CRISPR-assistant detection (ExCad), a simple, rapid, accurate gene detection tool for unextracted colonies and samples. We established a pretreatment protocol to rapidly liquify sputum samples and release nucleic acids within 10 min. The ExCad results can be visualised by a real-time fluorescence reader or the naked eye under blue light. We developed an ExCad-Sp assay to detect Streptococcus pneumoniae from unextracted strains and specimens, and optimised the assay conditions. Assay feasibility was evaluated using sputum samples from 32 patients, and it achieved 92.9 % (13/14) sensitivity, 100 % (18/18) specificity, 100 % (13/13) positive predictive value, and 94.7 % (18/19) negative predictive value compared with bacteria culture. The ExCad-Sp assay has potential for developing an at-home self-testing kit for S. pneumoniae.

CDetection.v2: One-pot assay for the detection of SARS-CoV-2.

Introduction: The ongoing 2019 coronavirus disease pandemic (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, is a global public health threat. Early diagnosis and identification of SARS-CoV-2 and its variants plays a critical role in COVID-19 prevention and control. Currently, the most widely used technique to detect SARS-CoV-2 is quantitative reverse transcription real-time quantitative PCR (RT-qPCR), which takes nearly 1 hour and should be performed by experienced personnel to ensure the accuracy of results. Therefore, the development of a nucleic acid detection kit with higher sensitivity, faster detection and greater accuracy is important. Methods: Here, we optimized the system components and reaction conditions of our previous detection approach by using RT-RAA and Cas12b. Results: We developed a Cas12b-assisted one-pot detection platform (CDetection.v2) that allows rapid detection of SARS-CoV-2 in 30 minutes. This platform was able to detect up to 5,000 copies/ml of SARS-CoV-2 without cross-reactivity with other viruses. Moreover, the sensitivity of this CRISPR system was comparable to that of RT-qPCR when tested on 120 clinical samples. Discussion: The CDetection.v2 provides a novel one-pot detection approach based on the integration of RT-RAA and CRISPR/Cas12b for detecting SARS-CoV-2 and screening of large-scale clinical samples, offering a more efficient strategy for detecting various types of viruses.

Visual detection of SARS-CoV-2 with a CRISPR/Cas12b-based platform

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, highlighting the unprecedented demand for rapid and portable diagnostic methods. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins-based platforms have been used for the detection of pathogens. However, in further applications and research, due to multiple steps needed, many methods showed an increased risk of cross-reactivity. The thermostable Cas12b enables the combination of isothermal amplification and CRISPR-mediated detection, which could decrease the risk of cross-contamination. In this study, we developed a portable and specific diagnostic method that combined the gold nanoparticle (AuNP) with thermal stable CRISPR/Cas12b-enhanced reverse transcription loop-mediated isothermal amplification (RT-LAMP), which is called SCAN, to distinguish the N gene of SARS-CoV-2 from flu gene. We validated our method using RNA from cells transfected by plasmids. We could easily distinguish the positive results by the naked eye based on the strong molar absorption coefficient of AuNP. Moreover, SCAN has the potential for high-throughput tests owing to its convenient operation. In sum, SCAN has broken the site and equipment restrictions of traditional detection methods and could be applied outside of hospitals and clinical laboratories, greatly expanding the test of COVID-19. [Display omitted] • The CRISPR/Cas12 b was employed to realize one-tube detection. • The SCAN assay is isothermal that requires minimal equipment. • The SCAN assay has a high-throughput potential for large-scale population screening. [ FROM AUTHOR]

Visual detection of SARS-CoV-2 with a CRISPR/Cas12b-based platform

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, highlighting the unprecedented demand for rapid and portable diagnostic methods. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins-based platforms have been used for the detection of pathogens. However, in further applications and research, due to multiple steps needed, many methods showed an increased risk of cross-reactivity. The thermostable Cas12 b enables the combination of isothermal amplification and CRISPR-mediated detection, which could decrease the risk of cross-contamination. In this study, we developed a portable and specific diagnostic method that combined the gold nanoparticle (AuNP) with thermal stable CRISPR/Cas12 b-enhanced reverse transcription loop-mediated isothermal amplification (RT-LAMP), which is called SCAN, to distinguish the N gene of SARS-CoV-2 from flu gene. We validated our method using RNA from cells transfected by plasmids. We could easily distinguish the positive results by the naked eye based on the strong molar absorption coefficient of AuNP. Moreover, SCAN has the potential for high-throughput tests owing to its convenient operation. In sum, SCAN has broken the site and equipment restrictions of traditional detection methods and could be applied outside of hospitals and clinical laboratories, greatly expanding the test of COVID-19.

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. 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.).

iSCAN-V2: A One-Pot RT-RPA-CRISPR/Cas12b Assay for Point-of-Care SARS-CoV-2 Detection.

Rapid, specific, and sensitive detection platforms are prerequisites for early pathogen detection to efficiently contain and control the spread of contagious diseases. Robust and portable point-of-care (POC) methods are indispensable for mass screening of SARS-CoV-2. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based nucleic acid detection technologies coupled with isothermal amplification methods provide a straightforward and easy-to-handle platform for detecting SARS-CoV-2 at POC, low-resource settings. Recently, we developed iSCAN, a two-pot system based on coupled loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a reactions. However, in two-pot systems, the tubes must be opened to conduct both reactions; two-pot systems thus have higher inherent risks of cross-contamination and a more cumbersome workflow. In this study, we developed and optimized iSCAN-V2, a one-pot reverse transcription-recombinase polymerase amplification (RT-RPA)-coupled CRISPR/Cas12b-based assay for SARS-CoV-2 detection, at a single temperature in less than an hour. Compared to Cas12a, Cas12b worked more efficiently in the iSCAN-V2 detection platform. We assessed and determined the critical factors, and present detailed guidelines and considerations for developing and establishing a one-pot assay. Clinical validation of our iSCAN-V2 detection module with reverse transcription-quantitative PCR (RT-qPCR) on patient samples showed 93.75% sensitivity and 100% specificity. Furthermore, we coupled our assay with a low-cost, commercially available fluorescence visualizer to enable its in-field deployment and use for SARS-CoV-2 detection. Taken together, our optimized iSCAN-V2 detection platform displays critical features of a POC molecular diagnostic device to enable mass-scale screening of SARS-CoV-2 in low-resource settings.

A one-step, one-pot CRISPR nucleic acid detection platform (CRISPR-top): Application for the diagnosis of COVID-19.

The existing CRISPR-mediated diagnostic tests require a two-step procedure (DNA or RNA amplification followed by CRISPR-mediated sequence-specific detection) for nucleic acid detection, which increases complexity and the risk of sample cross-contamination. Here, we report a new CRISPR-mediated test, called CRISPR-top (CRISPR-mediated testing in one-pot), which integrates simultaneous target pre-amplification with CRISPR/cas12b-mediated detection into a one-pot reaction mixture, performed at a constant temperature. The novel CRISPR-top assay was applied to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19 (coronavirus disease 2019). COVID-19 CRISPR-top targets the ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2, and operates at 59 °C for 40 min with minimal instrument. The COVID-19 CRISPR-top assay can return results within 60-min and is easily interpreted by visual fluorescence or lateral flow readouts. The analytical limit of detection (LoD) for COVID-19 CRISPR-top is 10 copies (for each detection target) per reaction with no cross-reactivity observed from non-SARS-CoV-2 templates. Among clinically collected non-COVID-19 samples, the assay's specificity was 100% (80/80 oropharynx swab samples). Among 52 COVID-19 positive clinical samples collected, the COVID-19 CRISPR-top assay yielded 38 (73.1%) positive results using fluorescence readout and 35 (67.3%) positive results with lateral-flow readout. These diagnostic results were similar to those obtained using RT-PCR (34 positive (65.4%)). These data indicate that COVID-19 CRISPR-top is a simple, rapid, accurate and highly sensitive method for SARS-CoV-2 detection which can be used in the clinic, field laboratories and primary care facilities in resource-challenged settings.