CRISPR-cas13 enzymology rapidly detects SARS-CoV-2 fragments in a clinical setting.

Abstract

BackgroundThe well-recognized genome editing ability of the CRISPR-Cas system has triggered significant advances in CRISPR diagnostics. This has prompted an interest in developing new biosensing applications for nucleic acid detection. Recently, such applications have been engineered for detection of SARS-CoV-2. Increased demand for testing and consumables of RT-PCR assays has led to the use of alternate testing options. Here we evaluate the accuracy and performance of a novel fluorescence-based assay that received EUA authorization for detecting SARS-CoV-2 in clinical samples.MethodsThe Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology forms the basis of the Sherlock CRISPR SARS-CoV-2 kit using the CRISPR-Cas13a system. Our experimental strategy included selection of COVID-19 patient samples from previously validated RT-PCR assays. Positive samples were selected based on a broad range of cycle thresholds.ResultsA total of 60 COVID-19 patient samples were correctly diagnosed with 100% detection accuracy (relative fluorescence ratios: N gene 95% CI 29.9–43.8, ORF1ab gene 95% CI 30.1–46.3). All controls, including RNase P, showed expected findings. Overall ratios were robustly distinct between positive and negative cases relative to the pre-established 5-fold change in fluorescence.ConclusionsWe have evaluated the accuracy of detecting conserved targets of SARS-CoV-2 across a range of viral loads, including low titers, using SHERLOCK CRISPR collateral detection in a clinical setting. These findings demonstrate encouraging results, at a time when COVID-19 clinical diagnosis and screening protocols remain in demand; especially as new variants emerge and vaccine mandates evolve. This approach highlights new thinking in infectious disease identification and can be expanded to measure nucleic acids in other clinical isolates.