A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern.
A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern.
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DOI：
10.1016/j.ebiom.2022.103926
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发表时间：
2022-03
期刊：
影响因子：
11.1
通讯作者：
Jain PK
中科院分区：
文献类型：
--
作者：
Nguyen LT;Macaluso NC;Pizzano BLM;Cash MN;Spacek J;Karasek J;Miller MR;Lednicky JA;Dinglasan RR;Salemi M;Jain PK
目前的SARS-CoV-2检测平台缺乏有效区分关注变体（voc）的能力。基于CRISPR/Cas（聚集规律间隔短回文重复序列/CRISPR相关）的检测系统由于其可编程性，有可能改变COVID-19诊断的格局；然而，这些方法大多依赖于涉及扩增或精心设计向导RNA的多步骤过程。分别从嗜酸地酸芽孢杆菌（AacCas12b）、嗜酸酸酸芽孢杆菌（AapCas12b）和短芽孢杆菌sp. SYP-B805 （BrCas12b）中表达和纯化了3个Cas12b蛋白，并通过差示扫描荧光法、顺式和反式剪切活性在一定温度范围内表征了它们的热稳定性。然后将BrCas12b纳入基于逆转录环介导的等温扩增（RT-LAMP）的单锅反应体系，称为CRISPR- spade （CRISPR Single Pot Assay for detection Emerging VOCs）。在这里，我们描述了一个完整的单锅检测反应，利用来自短芽孢杆菌的耐热Cas12b效应核酸内切酶来克服这些挑战，检测和区分临床样品中的SARS-CoV-2挥发性有机化合物。利用CRISPR-SPADE对Alpha （B.1.1.7）、Beta （B.1.351）、Gamma （P.1）、Delta （B.1.617.2）和Omicron （B.1.1.529）等SARS-CoV-2 VOCs进行识别，并在208份临床样本中进行验证。CRISPR-SPADE在10-30 min内鉴别SARS-CoV-2 VOCs的灵敏度为92.8%，特异性为99.4%，准确性为96.7%，与S基因测序结果一致，阳性预测值为99.1%，阴性预测值为95.1%。有趣的是，对于病毒载量高的样本（Ct值≤30），准确度和灵敏度达到100%。为了促进该分析的传播和全球实施，开发了一种冻干版的单锅CRISPR-SPADE试剂，并与能够解释两个正交荧光信号的内部便携式多路复用设备相结合。该技术能够实时监测rt - lamp介导的扩增和基于crispr的反应，成本仅为qPCR系统的一小部分。耐热短芽孢杆菌sp. Cas12b提供了轻松的引物设计，可以在简单可靠的单锅试验中准确检测SARS-CoV-2挥发性有机物。冻干试剂和简单仪器进一步实现了可快速部署的即时诊断，可轻松扩展到COVID-19之外。
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. 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). 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. 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.
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影响因子：
33.5
作者：
Teng, Fei;Cui, Tongtong;Li, Wei
通讯作者：
Li, Wei
DOI：
10.1038/s41577-021-00592-1
发表时间：
2021-10
期刊：
Nature reviews. Immunology
影响因子：
--
作者：
Tregoning JS;Flight KE;Higham SL;Wang Z;Pierce BF
通讯作者：
Pierce BF
影响因子：
3.7
作者：
Bhoyar RC;Jain A;Sehgal P;Divakar MK;Sharma D;Imran M;Jolly B;Ranjan G;Rophina M;Sharma S;Siwach S;Pandhare K;Sahoo S;Sahoo M;Nayak A;Mohanty JN;Das J;Bhandari S;Mathur SK;Kumar A;Sahlot R;Rojarani P;Lakshmi JV;Surekha A;Sekhar PC;Mahajan S;Masih S;Singh P;Kumar V;Jose B;Mahajan V;Gupta V;Gupta R;Arumugam P;Singh A;Nandy A;P V R;Jha RM;Kumari A;Gandotra S;Rao V;Faruq M;Kumar S;Reshma G B;Varma G N;Roy SS;Sengupta A;Chattopadhyay S;Singhal K;Pradhan S;Jha D;Naushin S;Wadhwa S;Tyagi N;Poojary M;Scaria V;Sivasubbu S
通讯作者：
Sivasubbu S
CRISPR-Cas12a辅助核酸检测
影响因子：
33.5
作者：
Li SY;Cheng QX;Wang JM;Li XY;Zhang ZL;Gao S;Cao RB;Zhao GP;Wang J
通讯作者：
Wang J
影响因子：
4.7
作者：
Li, Linxian;Li, Shiyuan;Wang, Jin
通讯作者：
Wang, Jin