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

BACKGROUND: The 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. METHODS: The 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. RESULTS: A 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. CONCLUSIONS: We 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.

High-Throughput CRISPR-Cas13 SARS-CoV-2 Test.

BACKGROUND: The ability to control the spread of COVID-19 continues to be hampered by a lack of rapid, scalable, and easily deployable diagnostic solutions. METHODS: We developed a diagnostic method based on CRISPR (clustered regularly interspaced short palindromic repeats) that can deliver sensitive, specific, and high-throughput detection of Sudden Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2). The assay utilizes SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for the qualitative detection of SARS-CoV-2 RNA and may be performed directly on a swab or saliva sample without nucleic acid extraction. The assay uses a 384-well format and provides results in <1 hour. RESULTS: Assay performance was evaluated with 105 (55 negative, 50 positive) remnant SARS-CoV-2 specimens previously tested using Food and Drug Administration emergency use authorized assays and retested with a modified version of the Centers for Disease Control and Prevention (CDC) quantitative PCR with reverse transcription (RT-qPCR) assay. When combined with magnetic bead-based extraction, the high-throughput SHERLOCK SARS-CoV-2 assay was 100% concordant (n = 60) with the CDC RT-qPCR. When used with direct sample addition the high-throughput assay was also 100% concordant with the CDC RT-qPCR direct method (n = 45). With direct saliva sample addition, the negative and positive percentage agreements were 100% (15/15, 95% CI: 81.8-100%) and 88% (15/17, 95% CI: 63.6-98.5%), respectively, compared with results from a collaborating clinical laboratory. CONCLUSIONS: This high-throughput assay identifies SARS-CoV-2 from patient samples with or without nucleic acid extraction with high concordance to RT-qPCR methods. This test enables high complexity laboratories to rapidly increase their testing capacities with simple equipment.