Rapid and sensitive detection of COVID-19 using CRISPR/Cas12a-based detection with naked eye readout, CRISPR/Cas12a-NER.

The outbreak of the 2019 novel coronavirus disease (COVID-19) has had a significant impact on global health. To advance the diagnostic method of COVID-19, we developed a CRISPR/Cas12a-based assay with a naked eye readout, CRISPR/Cas12a-NER. The assay can detect as few as 10 copies of the virus gene in 45 min without a special instrument and has good consistency with the qPCR assay, providing a simple and reliable on-site diagnostic method suitable for a local hospital or community testing.

The novel coronavirus disease (COVID-19) had caused more than 1,991,562 confirmed cases with 130,885 deaths by 16 April 2020 (World Health Organization Coronavirus disease 2019 situation reports). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to the disease [1]. SARS-CoV-2, a positive-sense virus, belongs to Betacoronavirus genus lineage β and is phylogenetically closely related to bat SARS-like coronaviruses [2].

Sensitive and rapid detection of COVID-19 is crucial in controlling the outbreak. Currently, real-time quantitative reverse transcription-polymerase chain reaction (qPCR) assays are recommended as molecular diagnostic methods [3], [4]. However, qPCR requires an elaborate instrument and professional operation, which limits its diagnostic application. Recently, a CRISPR/Cas-based nucleic acid detection technology was developed with the advantages of sensitivity, specificity, rapidity, and simplicity [5], [6]. To advance the on-site diagnosis of COVID-19, we introduce a CRISPR/Cas12a-based rapid detection assay.

To sensitively and rapidly detect SARS-CoV-2 nucleic acid, CRISPR/Cas12a-based-detection with naked eye readout, termed CRISPR/Cas12a-NER, including the Cas12a protein, SARS-CoV-2 specific CRISPR RNAs (crRNAs) and a single-stranded DNA (ssDNA) reporter, was developed as shown in Fig. 1 a. To enable on-site diagnosis, an ssDNA reporter labelled with a quenched green fluorescent molecule was introduced, which will be cleaved by Cas12a when there is nucleic acid of SARS-CoV-2 in the detection system, and the resulting green fluorescence can be seen with the naked eye under 485 nm light (Fig. 1a).

Fig. 1

CRISPR/Cas12a-based detection with naked eye readout (CRISPR/Cas12a-NER) of SARS-CoV-2. (a) Schematic of Cas12a-based assay for rapid and visual nucleic acid detection. After 30 min of RT-RAA reaction, the Cas12a enzyme targets cleavage for an additional 15 min. The green fluorescent signals can be observed by the naked eye under blue light with a wavelength of 485 nm. (b) Four target gene fragments and SARS-CoV-2-specific crRNAs location on the genome. (c) Validation of crRNA specifically reacts with the target gene. (d) Time course of E gene detection with E-crRNAmix on gradient diluted input RNAs from 1e4 to 1e0 copies. (e) Sensitivity assay using E-crRNAmix to detect gradient diluted E gene RNA from 1e4 to 1e0 copies. (f) Diagnostic accuracy of CRISPR/Cas12a-NER on the diagnosis of COVID-19 clinical samples. ***: statistical significance; NS: not significance.

For specific detection of the SARS-CoV-2 genome, a total of 15 crRNAs, which are predicted to distinguish single nucleotide polymorphisms (SNPs) with other SARS or SARS-related viruses, were designed on four domains of the orf1a, orf1b, N and E genes over the Wuhan-Hu-1 strain (GenBank accession number MN908947) (Fig. 1b, Fig. S1 online). The results showed that 14 crRNAs, except the E-crRNA1, targeting SARS-CoV-2 were validated, and all of them highly and specifically reacted with synthetic gene fragments (Fig. 1c, Figs. S2 and S3 online). As the CRISPR/Cas12a-NER readout signal relies on the crRNA-dependent targeting cleavage efficiency, which is affected by the secondary structure and spacer sequence of the crRNA [7], [8]. The crRNAmix (equal mix work crRNAs targeting at each gene, named orf1a-crRNAmix, orf1b-crRNAmix, N-crRNAmix, E-crRNAmix) mediated the strongest fluorescence signal (Fig. S4 online) and was chosen for the detection of COVID-19.

As SARS-CoV-2 is an RNA virus, reverse transcript recombinase-aided amplification (RT-RAA) was combined to obtain enough DNA for Cas12a-mediated detection. The RT-RAA will amplify the target gene fragment in 30 min at 39 °C, followed by a CRISPR/Cas12a reaction at 37 °C (Fig. 1a). Cas12a-mediated detection produces a robust signal in a 15 min reaction (Fig. 1d), and then the assay time is fixed to 15 min. The sensitivity of the CRISPR/Cas12a-NER assay was determined by the fluorescence intensities. The results show that among the four target genes, the E-crRNAmix for the target E gene is the most sensitive, which can clearly detect 10 copies of SARS-CoV-2 synthetic nucleic acid (Fig. 1e, Fig. S5 online). Thus, we chose E-crRNAmix for a screening assay of SARS-CoV-2, and the remaining target genes can be used for a further confirmed diagnostic assay.

To validate the CRISPR/Cas12a-NER diagnostic accuracy, a total of 31 clinically extracted RNA samples were used for COVID-19 detection with the CRISPR/Cas12a-NER assay and TaqMan qPCR (GenScript, Nanjing, China) for the E gene in parallel. Clinical samples used in this study were collected and treated in strict accordance with the standard operation for COVID-19 recommended by the World Health Organization. All sample treatments were conducted in the State Key Laboratory of Respiratory Disease and Kingmed Virology Diagnostic & Translational Center (Guangzhou). Among these samples, consistent with clinical diagnosis, 16 were determined to be SARS-CoV-2 positive by both CRISPR/Cas12a-NER and qPCR assays and showed 100% agreement (Fig. 1f, Figs. S6 and S7 online), with the kappa value (κ) being 1.0 (P < 0.001) (Table S3 online), confirming the outstanding performance of CRISPR/Cas12a-NER.

Overall, we described CRISPR/Cas12a-NER, which provides a novel alternative for the portable, simple, sensitive, and specific detection of the COVID-19 virus, providing a simple and reliable on-site diagnostic method suitable for a local hospital or community testing.

Conflict of interest

The author declares that they have no conflict of interest.