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

Abstract

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 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 less than one hour. RESULTS Assay performance was evaluated with 105 (55 negative, 50 positive) remnant SARS-CoV-2 specimens previously identified as positive using Food and Drug Administration emergency use authorized assays and re-tested with a modified version of the Centers for Disease Control and Prevention (CDC) 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 percent 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.

Smartphone-read phage lateral flow assay for point-of-care detection of infection

“…Such leftover clinical samples are typically used in the initial evaluation of a test under development when available and affordable to purchase. 5,31,32 Studies with these de-identified clinical specimens were not considered human subjects research. Handling and testing of clinical specimens were performed under BSL-2 containment inside a Nuaire Class II Biological Safety Cabinet (UH MUA #008-20).…”

Evaluation of an automated CRISPR-based diagnostic tool for rapid detection of COVID-19

“…Their test exhibits a specificity and positive predictive value of 100% and required less time (within 30 min) to obtain restuls [ 40 ]. Li et al published an enhanced strip method based on CRISPR, which was used to visualize the results directly and was 100% concordant with the RT-PCR method [ 41 ]. Finally, Hou et al reported a 100% sensitivity for the diagnostic performance of ‘CRISPR-nCoV’ for SARS-CoV-2 detection [ 42 ].…”

CRISPR-Cas13 in malaria parasite: Diagnosis and prospective gene function identification

“…To improve signal generation, Yang et al (2022) engineered a LwCas13a with improved collateral activity by inserting different RNA-binding domains into a unique site within the HEPN domain. The SHERLOCK application has been developed into a cheap and convenient paper-based CRISPR-Cas13-base diagnosing assay capable of detecting and differentiating RNAs of viruses (Zika virus and Dengue virus) and genotyping DNA of bacteria ( Escherichia coli and Pseudomonas aeruginosa ; Gootenberg et al, 2017 ; Myhrvold et al, 2018 ; Manning et al, 2021 ).…”