Simplified Cas13-based assays for the fast identification of SARS-CoV-2 and its variants

Abstract

The widespread transmission and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) call for rapid nucleic acid diagnostics that are easy to use outside of centralized clinical laboratories. Here we report the development and performance benchmarking of Cas13-based nucleic acid assays leveraging lyophilised reagents and fast sample inactivation at ambient temperature. The assays, which we named SHINEv.2 (for 'streamlined highlighting of infections to navigate epidemics, version 2'), simplify the previously reported RNA-extraction-free SHINEv.1 technology by eliminating heating steps and the need for cold storage of the reagents. SHINEv.2 detected SARS-CoV-2 in nasopharyngeal samples with 90.5% sensitivity and 100% specificity (benchmarked against the reverse transcription quantitative polymerase chain reaction) in less than 90 min, using lateral-flow technology and incubation in a heat block at 37 °C. SHINEv.2 also allows for the visual discrimination of the Alpha, Beta, Gamma, Delta and Omicron SARS-CoV-2 variants, and can be run without performance losses by using body heat. Accurate, easy-to-use and equipment-free nucleic acid assays could facilitate wider testing for SARS-CoV-2 and other pathogens in point-of-care and at-home settings.

Extended Data Fig. 1 ∣. SHINEv.2 requires 5 user manipulations.

Schematic of the SHINEv.2 workflow, including sample inactivation, rehydration of pellets, sample addition, reaction dilution after incubation and result readout.

Extended Data Fig. 2 ∣. Performance of SARS-CoV-2 Delta and Omicron assays on synthetic RNA targets.

SHINE fluorescence of the (a) 452, (b) 156 - 158 and (c) 142 - 145 ancestral (anc) and derived (der) assays on (a,b) full genome synthetic RNA standards or (c) synthetic RNA targets after 90 minutes. NTC, no target control. Center = mean and error bars = s.d. for 3 technical replicates.

Extended Data Fig. 3 ∣. Validation of SHINEv.2 Delta assays.

a, Discrimination of SARS-CoV-2 VOCs using SHINE fluorescence of Delta assays on full-genome synthetic RNA standards, after 90 minutes. Target RNA concentration: 104 genomes/μL. (b,c), Colorimetric lateral flow based detection of full-genome synthetic RNA standards using the (b) 156 - 158 and (c) 452 SHINEv.2 assays. SHINEv.2 incubation time: 90 minutes. NTC, no target control. T, test line; C, control line.

Extended Data Fig. 4 ∣. Development of Cas12-based SHINE assay for RNase P detection.

a. SHINE fluorescence of the RNase P assay on synthetic DNA target after 90 minutes. b. Lateral flow detection of synthetic DNA target using Cas12-based SHINE assay, after 90 minute incubation; c. SHINE fluorescence of the duplex SARS-CoV-2 S gene and human RNase P assays on synthetic nucleic acid targets after 90 minutes. NTC = no target control; C = control band; T = test band. For (a,c), center = mean and error bars = s.d. for 3 technical replicates.

Fig. 1 ∣. Increasing the ease-of-use and deployability of SHINE.

a, RNase activity in nasal fluid mixed with UTM untreated or treated with FastAmp lysis reagent supplemented with RNase inhibitor (inh.) or treated with HUDSON (a heat- and chemical-treatment). Activity measured using RNaseAlert at r.t. for 30 min. b, SARS-CoV-2 seedstock titre without treatment or after being incubated with lysis reagent (+5% RNase inh.) at r.t. for 5 min, 20 min or 20 min+10 min at 65 °C. ***, infection not detected; PFU, plaque forming units. c, SHINE fluorescence with different proportions of blank inactivated sample input (that is, FastAmp lysis reagent, RNase inh. and UTM) after a 90 min incubation. Base*, baseline (that is, no inactivated sample added). d, Schematic of the advantages of lyophilising SHINE. e, SHINE fluorescence on synthetic RNA target (10⁴ cp μl⁻¹) before and after lyophilisation using different buffers. Fluorescence measured after 90 min. For buffer composition, see Methods. f, SHINE fluorescence after a 90 min incubation using lyophilised (LYO) reagents stored at r.t., 4 °C or −20 °C over time. Target concentration: 10⁴ cp μl⁻¹. g, Fluorescence kinetics for SHINEv.1 and SHINEv.2 using synthetic RNA targets; NTC, no target control. h, Lateral-flow detection of SARS-CoV-2 RNA in lysis buffer-treated viral seedstocks using SHINEv.2 after a 90 min incubation. C, control band; T, test band. i, Determination of analytical limit of detection with 20 replicates of SHINEv.2 at different concentrations of SARS-CoV-2 RNA from lysis reagent-treated contrived samples incubated for 90 min. Mean ± s.d. for 3 technical replicates for a, e and g; 2 technical replicates for b; 3 biological replicates with 3 technical replicates each for f. In c, heatmap values are the means of 3 technical replicates.

Fig. 2 ∣. Performance of SHINEv.2 on clinical samples.

a, Positive and negative SHINEv.2 test results for RT-qPCR-positive and negative clinical samples relative to viral RNA concentration. b, Concordance between SHINEv.2 and RT-qPCR for 72 nasopharyngeal swab samples. c, Schematic of side-by-side clinical sample testing using SHINEv.2, BinaxNow, CareStart and RT-qPCR. NP, nasopharyngeal swab. d, Positive and negative test results for SHINEv.2, BinaxNow and CareStart tests for RT-qPCR-positive clinical samples relative to viral RNA concentration.

Fig. 3 ∣. Development of SHINEv.2 assays for the detection of SARS-CoV-2 VOCs.

a, Schematic of Cas13a-based detection of mutations in SARS-CoV-2 using a fluorescent readout; anc, ancestral; der, derived. b,c, SHINE fluorescence of the ancestral and derived crRNAs for the 69/70 deletion assay (b) and the 417 mutation assay (c) on synthetic RNA targets after 90 min; der N, derived N (K417N); der T, derived T (K417T). d, Identification of SARS-CoV-2 VOCs using SHINE fluorescence on full-genome synthetic RNA standards and RNA extracted from viral seedstocks; target RNA concentration: 10⁴ cp μl⁻¹. e, Mean fluorescence of 69/70 SHINEv.2 assay on SARS-CoV-2 RNA extracted from clinical samples after 90 min. f, Lateral-flow detection of SARS-CoV-2 RNA and VOC discrimination with SHINEv.2 Omicron assay on unextracted clinical samples after a 90 min incubation. RT-qPCR cycle threshold (C_t) specified for each sample; –, no amplification detected (SARS-CoV-2-negative sample). For b and c, mean ± s.d. for 3 technical replicates. In d, e and f, the heatmap values represent the mean for 3 technical replicates; *, positive signal expected.

Fig. 4 ∣. Enhancing the accessibility of SHINEv.2.

a,b, RNase P SHINEv.2 assay results on synthetic DNA target after 90 min using a plate-based readout (a) and a lateral-flow-based readout (b). c, Lateral-flow-based detection of SARS-CoV-2 RNA using SHINEv.2 with different PEG compositions with or without dilution after a 90 min incubation. d, Lateral-flow-based SHINEv.2 detection of SARS-CoV-2 RNA after a 90 min incubation in a heat block or using body heat (underarm incubation). e, Determination of analytical LoD with 20 replicates of SHINEv.2 at 200 cp μl⁻¹ of SARS-CoV-2 RNA from lysis solution-treated contrived samples incubated for 90 min. f, SHINE fluorescence on SARS-CoV-2 RNA after 90 min at 37°C or 25°C. For a and f, mean ± s.d. for 3 technical replicates.