Clinical validation of engineered CRISPR/Cas12a for rapid SARS-CoV-2 detection

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.