Nguyen Long T, Macaluso Nicolas C, Pizzano Brianna L M, Cash Melanie N, Spacek Jan, Karasek Jan, Dinglasan Rhoel R, Salemi Marco, Jain Piyush K
medRxiv. 2021 Oct 18:2021.10.15.21265066. doi: 10.1101/2021.10.15.21265066.
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 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 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 (C 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.
目前的严重急性呼吸综合征冠状病毒2（SARS-CoV-2）检测平台缺乏以有效方式区分关注变体（VOCs）的能力。由于其可编程性，CRISPR（成簇规律间隔短回文重复序列）有潜力变革诊断方法。然而，许多基于CRISPR的检测方法依赖于涉及扩增的多步骤过程或精心设计的引导RNA。有人提出使用替代的Cas效应核酸酶进行完整的一锅法检测反应来克服这些挑战。然而，目前使用Cas12b（AapCas12b）的方法受到其在最佳逆转录环介导等温扩增（RT-LAMP）反应温度下热稳定性的限制。在此，我们证明来自sp. SYP-B805的一种新型Cas12b（称为BrCas12b）在理想的RT-LAMP条件下具有强大的反式切割活性。一项针对来自其他细菌属的Cas12b同源物的BrCas12b竞争性分析研究强调了BrCas12b在开发新诊断方法方面的潜力。作为概念验证，我们将BrCas12b纳入RT-LAMP介导的一锅法反应系统，命名为CRISPR-SPADE（用于检测新兴VOCs的CRISPR单锅检测法），以实现对SARS-CoV-2 VOCs的快速、差异检测，包括205份临床样本中的阿尔法（B.1.1.7）、贝塔（B.1.351）、伽马（P.1）和德尔塔（B.1.617.2）变体。值得注意的是，在扩增1 - 3分钟内观察到BrCas12b检测信号，在30分钟内总体特异性达到98.1%、准确率达到91.2%、灵敏度达到88.1%。重要的是，对于病毒载量高（C值≤30）的样本，准确率和灵敏度均达到100%。为了促进该检测方法的传播和全球应用，我们将冻干的一锅法试剂与能够以qPCR系统成本的一小部分解读荧光信号的便携式多路复用设备相结合。由于设计要求宽松、一锅法检测且仪器简单，该检测方法有能力推动未来的诊断发展。
