Clinical validation of engineered CRISPR/Cas12a for rapid SARS-CoV-2 detection.
Clinical validation of engineered CRISPR/Cas12a for rapid SARS-CoV-2 detection.
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DOI：
10.1038/s43856-021-00066-4
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Jain, Piyush K
中科院分区：
文献类型：
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作者：
Nguyen, Long T;Rananaware, Santosh R;Pizzano, Brianna L M;Stone, Brandon T;Jain, Piyush K
由SARS-CoV-2引起的冠状病毒病（COVID-19）以前所未有的速度席卷地球仪。基于CRISPR的检测技术已成为一种快速且经济实惠的平台，可以塑造诊断的未来。我们开发了ENHANCEv 2，它由嵌合向导RNA、修饰的LbCas 12 a酶和双报告基因构建体组成，以改善先前报道的ENHANCE系统。我们使用62个鼻咽拭子验证了ENHANCE和ENHANCEv 2，并将结果与RT-qPCR进行了比较。我们创建了ENHANCEv 2的冻干版本，并表征了其检测能力和稳定性。在这里，我们证明了当与RT-LAMP步骤结合时，ENHANCE以95%的准确度检测COVID-19样本，同时对31种高度相似和常见的呼吸道病原体的SARS-CoV-2的各种分离株保持高度特异性。ENHANCE在广泛的镁浓度范围内（3 mM-13 mM）稳定工作，允许进一步优化测定。我们对ENHANCE和ENHANCEv 2的临床验证结果显示，60/62（96.7%）的样品与RT-qPCR结果一致，而仅使用5 μL样品和20分钟的CRISPR反应。我们表明，使用纸基条带的侧流检测显示100%的协议与基于荧光的报告基因检测在临床验证。最后，我们证明了冻干版本的ENHANCEv 2对SARS-CoV-2检测显示出高灵敏度和特异性，同时将CRISPR反应时间缩短至3分钟，同时在室温下储存数周后保持其检测能力。与传统的基于qPCR的检测方法相比，基于CRISPR的诊断平台具有许多优势。我们的工作为ENHANCE及其改进形式ENHANCEv 2检测COVID-19提供了临床验证。Nguyen等人描述了ENHANCE系统的临床验证，该系统是一种基于Cas 12 a的工程化crRNA并在RT-LAMP扩增步骤之前检测SARS-CoV-2的方法。作者还描述了该系统的一个版本ENHANCEv 2的开发和临床验证，该系统可以冻干，并使用另一种突变的Cas 12 a进行进一步的信号放大。COVID-19大流行强调了快速准确检测SARS-CoV-2感染的必要性。通常使用的测试有局限性，基于CRISPR技术的检测系统可以提供一个有用的替代方案。CRISPR是一种源自细菌的技术，可以特异性地检测DNA片段。我们之前已经开发了ENHANCE，这是一种检测系统，可以将SARS-CoV-2遗传物质转化为DNA，然后通过工程CRISPR技术进行检测。在这里，我们开发了这种方法的改进版本ENHANCEv 2，它具有延长的保质期和更少的冷藏需求，便于测试及其使用所需组件的运输。我们发现ENHANCE和ENHACEv 2都可以快速准确地检测感染者拭子中的SARS CoV-2。这是朝着拥有更通用的工具来快速准确地检测SARS-CoV-2感染迈出的一步。
The coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies have emerged as a rapid and affordable platform that can shape the future of diagnostics. We developed ENHANCEv2 that is composed of a chimeric guide RNA, a modified LbCas12a enzyme, and a dual reporter construct to improve the previously reported ENHANCE system. We validated both ENHANCE and ENHANCEv2 using 62 nasopharyngeal swabs and compared the results to RT-qPCR. We created a lyophilized version of ENHANCEv2 and characterized its detection capability and stability. Here we demonstrate that when coupled with an RT-LAMP step, ENHANCE detects COVID-19 samples down to a few copies with 95% accuracy while maintaining a high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. ENHANCE works robustly in a wide range of magnesium concentrations (3 mM-13 mM), allowing for further assay optimization. Our clinical validation results for both ENHANCE and ENHANCEv2 show 60/62 (96.7%) sample agreement with RT-qPCR results while only using 5 µL of sample and 20 minutes of CRISPR reaction. We show that the lateral flow assay using paper-based strips displays 100% agreement with the fluorescence-based reporter assay during clinical validation. Finally, we demonstrate that a lyophilized version of ENHANCEv2 shows high sensitivity and specificity for SARS-CoV-2 detection while reducing the CRISPR reaction time to as low as 3 minutes while maintaining its detection capability for several weeks upon storage at room temperature. CRISPR-based diagnostic platforms offer many advantages as compared to conventional qPCR-based detection methods. Our work here provides clinical validation of ENHANCE and its improved form ENHANCEv2 for the detection of COVID-19. Nguyen et al. describe the clinical validation of the ENHANCE system, a method to detect SARS-CoV-2 based on engineered crRNAs for Cas12a and preceded by an RT-LAMP amplification step. Authors also describe the development and clinical validation of a version of this system, ENHANCEv2, that can be lyophilized and that uses another mutated Cas12a for further signal amplification. The COVID-19 pandemic has underscored the need for rapid and accurate tests to detect SARS-CoV-2 infection. The tests commonly used have limitations, and a detection system based on CRISPR technology could offer a useful alternative. CRISPR is a technology derived from bacteria that can specifically detect pieces of DNA. We have previously developed ENHANCE, a detection system that converts the SARS-CoV-2 genetic material into DNA that is then detected by an engineered CRISPR technology. Here, we develop an improved version of this method, ENHANCEv2, that has an extended shelf life and less need for refrigeration, facilitating transportation of the components required for the test and its use. We show that both ENHANCE and ENHACEv2 can quickly and accurately detect SARS-CoV-2 in swabs from infected people. This is a step towards having more versatile tools to detect SARS-CoV-2 infection quickly and accurately.