CRISPR-Cas12-based detection of SARS-CoV-2

Abstract

An outbreak of betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with SARS-CoV-2 infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT-PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.

Figure 1.

A CRISPR-Cas12 based assay for detection of SARS-CoV-2. (a) Genome map showing primers, probes and gRNAs. Visualization of primers and probes on the SARS-CoV-2 genome. RT-LAMP primers are indicated by black rectangles, the binding position of the F1c and B1c half of the FIP primer (grey) is represented by a striped rectangle with dashed borders. (b) gRNA specificity. Cas12 gRNAs are programmed to specifically target SARS-CoV-2 or broadly detect related coronavirus strains. The N gene gRNA used in the assay (left) is specific for SARS-CoV-2, whereas the E gene gRNA is able to detect 3 SARS-like coronavirus strains (right). A separate N gene gRNA designed to target SARS-CoV and a bat coronavirus fails to detect SARS-CoV-2 (middle). (c) Minimum equipment needed to run protocol. With appropriate biosafety level 2 requirements, the minimum equipment required to run the protocol includes Eppendorf tubes with reagents, heat blocks or water bath (37°C and 62°C), nuclease-free water, pipettes and tips, and lateral flow strips. (d) Schematic of SARS-CoV-2 DETECTR workflow. Conventional RNA extraction can be used as an input to DETECTR (LAMP pre-amplification and Cas12-based detection for E gene, N gene and RNase P), which is visualized by a fluorescent reader or lateral flow strip. (e) Lateral flow strip assay readout. A positive result requires detection of at least one of the two SARS-CoV-2 viral gene targets (N gene or E gene, as indicated in the interpretation matrix).

Figure 2.

Detection of SARS-CoV-2 in contrived and clinical nasopharyngeal or oropharyngeal swab samples. (a) Schematic of DETECTR coupled with lateral flow readout. The intact FAM-biotinylated reporter molecule flows to the control capture line. Upon recognition of the matching target, the Cas-gRNA complex cleaves the reporter molecule, which flows to the target capture line. (b-c) Comparison of fluorescence to lateral flow. (b) Fluorescence signal of LbCas12a detection assay on RT-LAMP amplicon for SARS-CoV-2 N gene saturates within 10 min. RT-LAMP amplicon generated from 2 µL of 5 fM or 0 fM SARS-CoV-2 N gene IVT RNA by amplifying at 62°C for 20 min. Error bars: mean±SD.(c) LbCas12a on the same RT-LAMP amplicon produces visible signal through lateral flow assay within 5 min. (d) Limit of detection for CDC qPCR and DETECTR. Ct values using the CDC qPCR assay (n=3) and fluorescence values using SARS-CoV-2 DETECTR (n=6) using SARS-CoV-2 N2 gene IVT-RNA. Representative lateral flow results for the assay shown for 0 copies/µL and 10 copies/µL. (e) Patient sample DETECTR data. Clinical samples from 6 patients with COVID-19 infection (n=11, 5 replicates) and 12 patients infected with influenza or one of the 4 seasonal coronaviruses (HCoV-229E, HCoV-HKU1, HCoV-NL63, HCoV-OC43) (n=12) were analyzed using SARS-CoV-2 DETECTR. Signal intensities from lateral flow strips were quantified using ImageJ and normalized to the highest value within the N gene, E gene or RNase P set, with a positive threshold set at five standard deviations above background. The final determination for SARS-CoV-2 test results was based on the interpretation matrix in Fig. 1e, with results indicated above the heat map. (f) Lateral flow strips showing SARS-CoV-2 DETECTR assay results. Two replicate assays were performed using 2 µL of extracted RNA for each reaction (titer 12 copies/µL). Positive controls used IVT RNA for SARS-CoV-2 targets and total human RNA for RNase P. LbCas12a detection assays were run on lateral flow strips (TwistDx) and imaged after 3 min. (g) Performance characteristics of the fluorescent SARS-CoV-2 DETECTR assay. 83 clinical samples (41 COVID-19 positive, 42 negative) were evaluated using the fluorescent version of the SARS-CoV-2 DETECTR assay (Supp. Fig. 7 a, c, d). One sample (COVID19–3) was omitted due to failing assay quality control. Positive and negative calls are based on criteria described in Fig. 1e. Abbreviations: fM, femtomolar; NTC, no-template control; FLUA, influenza A virus; FLUB, influenza B virus; HCoV, human coronavirus; PPA, positive predictive agreement; NPA, negative predictive agreement.