CRISPR and Cas systems recognize and degrade foreign nucleic acid under the guidance of a sequence-specific RNA molecule. Some Cas enzymes like Cas12a, Cas12b, Cas13a, Cas13b, and Cas14 display collateral cleavage activity after binding to their specific targets, which has been fully evaluated for diagnostic use to detect nucleic acids. Upon target recognition, the activated Cas nucleases indiscriminately cleave nearby single-stranded DNA (ssDNA). By introducing an ssDNA reporter labeled with a fluorophore and quencher or alternatively with a fluorophore and biotin into an in vitro reaction, the cleavage could be detected with a fluorescent reader or a lateral flow paper strip.

Although all of these proteins are RNA-guided nucleases, Cas13a and Cas13b are RNases, while Cas12a, Cas12b and Cas14 are DNases. Therefore, the Cas13a- and Cas13b-based detections require a transcription step by RNA polymerase and, in contrast, Cas12a, Cas12b, and Cas14 can directly use DNA as substrates. The pre-amplification of nucleic acid with an isothermal or PCR method is normally required to enhance the detection sensitivity. By combining with the recombinase polymerase amplification (RPA) technology, Cas12a and Cas13a have both been shown to permit single-molecule detection in a given reaction.

In this study, we present an approach for the visual detection of the ORF1ab and N genes of SARS-CoV-2 using CRISPR-Cas12a system. By combination of a sample processing method and the RPA technology with Cas12a-based detection, the sample to result can be obtained in 50 minutes with a fluorescent reader or via the naked eye (Fig 1A).

Fig 1. Workflow of Cas12a-based detection for SARS-CoV-2.

Extracted RNA or lysed samples can be used as an input for RPA-Cas12a-based detection. (a) The results can be achieved within 50 minutes using a fluorescent reader or with visual detection methods. (b) The minimum equipment needed to run the assay following RNA extraction or rapid sample lysis includes UV light imagers, heat blocks (42°C and 37°C), pipettes and tips, Eppendorf tubes, reagents, and lateral flow strips. Cas, CRISPR associated proteins; RPA, recombinase polymerase amplification; RT, room temperature; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; UV, ultraviolet.

Results and discussion

As the sequences of oligonucleotides are crucial to a rapid and sensitive RPA, primers need to be screened to establish a preferred detection method. We tested 18 and 15 primer combinations for the target genes of ORF1ab and N, respectively, using 100 copies of RNA for each reaction. To screen out the best primer set for the ORF1ab gene, a reverse primer (R2) was used to against forward primers (F1 to F5), selecting the best forward primer (F5) and then using it to screen all the reverse primers (R1 to R5). The primer pair with the best performance was identified and used in subsequent experiments (F5+R1, Fig 2A–2D). Primers for the N gene of SARS-CoV-2 were subjected to the same procedure, and the primer set of F4+R2 exhibited the best performance (Fig 2E–2H).

Fig 2. Primer screening.

Primers for ORF1ab (a–d) and N (e–h) were screened using a single forward or reverse primer against the corresponding reverse or forward primers, selecting the best performing primer and then using it to perform second round screening to determine the primer pairs with the best performance. Fluorescent signal was obtained at 10 minutes for the Cas12a reaction.

To determine the analytical sensitivity of this system, the targets were serially diluted to 1, 10, and 100 copies/μL. Then, 1 μL of RNA from each dilution was used as template, and 3 independent replicates each with 5 technical replicates were performed. The CRISPR-Cas12a assays for ORF1ab and N gene were both capable of detecting their corresponding targets at all assayed dilutions and did not show any signals in the no-template control. These results illustrate our developed CRISPR-Cas12a assay has a single-copy sensitivity.

Serial dilutions of in vitro–transcribed ORF1ab and N for LoD determination. After amplification at 42°C for 20 minutes, 10 μL of each reaction was transferred to 40 μL of the Cas12a mixture for cleavage assays. Bar graphs represent fluorescent signal obtained at 10 minutes for the Cas12a reaction. The locations of primers and crRNAs of ORF1ab (upper) and N (lower) from 4 SARS-CoV-2 strains and 4 other human coronaviruses. Sequences of different types of coronavirus were obtained from GenBank and aligned by the MEGA program. Specificity assessment of the RT-RPA assay for ORF1ab gene and N gene of SARS-CoV-2. Only the RNA from SARS-CoV-2 produced signals, whereas RNAs from other pathogens and the negative control did not produce any detected signals. Four human coronaviruses (SARS, HKU1, MERS, and OC43) and 11 other respiratory pathogens including avian influenza A viruses (H7N9 and H5N1), human influenza A viruses (H1N1 [swine flu] and H3N2 subtypes), influenza B viruses (Yamagata and Victoria lineages), adenovirus (ADV3, 7), human parainfluenza virus (PIV2), Staphylococcus aureus, and Streptococcus pneumonia were evaluated.

We then aligned the primers with other coronavirus genomes (Human Coronavirus OC43 [HCoV-OC43], Human Coronavirus HKU1 [HCoV-HKU1], Severe Acute Respiratory Syndrome [SARS], and Middle East Respiratory Syndrome Coronavirus [MERS-CoV]) using MEGA 7 and found the sequences were not similar to most viral sequences (Fig 3C). Moreover, the crRNAs targeting the ORF1ab and N genes of SARS-CoV-2 have several mismatches to other coronavirus, suggesting high specificity of the assay to targets since crRNAs have been shown to be sensitive to single-nucleotide mutations. These results together demonstrate that the established system is highly specific to SARS-CoV-2.

Next, to mimic clinical situations, we spiked pseudovirus containing fragments of ORF1ab and N genes into samples from healthy volunteers. As compared to a live virus, pseudoviruses, which can be produced naturally during the infection or artificially by researchers in laboratories, contain nucleic acid sequences of interest but do not have any of the components for infectious viruses. We tested various protocols for sample lysis and found that the one that used Triton X-100, Tween-20, guanidine hydrochloride, and a heating step yielded the strongest signal. Nevertheless, the signal was significantly reduced when the heating step was abolished, implying incomplete sample lysis without heating. This protocol allowed the sample processing to be finished in 5 minutes, and the unextracted lysate could be directly served as templates for RT-RPA amplification. Our assay was able to detect a single copy of pseudovirus in a reaction.

Visual detection is crucial for nucleic acid diagnostics, especially in situations when instruments are not available. In this study, we used a reporter with a dye label (FAM) on the 5′ end and a fluorescent quencher (BHQ1) on the 3′ end. After the Cas12a cleavage assay, we observed bright green signals by the naked eye under a gel imager when the targets were present. With this method, a detection as sensitive as 1 copy per reaction was realized, which is comparable with the method using a fluorescence plate reader. We developed another reporter labeled with a FAM molecule on the 5′ end and a biotin on the 3′ end. When coupled with a lateral flow strip, the destruction of the reporter is visible by the naked eye. This method exhibited an analytical sensitivity of 1 copy/μL for the template.

We next compared the sensitivity of the CRISPR-Cas12a-based method with the RPA-only method, the latter of which appeared not to be as sensitive as the former with observed Limit of Detection (LoD) values of 50 copies/μL and 10 copies/μL for the ORF1ab and N genes, respectively, in lateral flow assay. In fluorescent detection assay, the RPA-only detection also showed an inferior sensitivity than Cas12a-based assay with LoD of 1,000 and 100 copies for ORF1ab and N genes, respectively, in a given reaction. This observation may ascribe to the signal amplification effect induced by the collateral activity of Cas12a. Therefore, it would be reasonable to speculate that the CRISPR-Cas12a-based detection approach is more sensitive than the RPA-only system in general.

Finally, we evaluated our system using 22 clinical samples initially diagnosed by RT-qPCR. The results showed 100% consistency between our assay and the RT-qPCR readouts for both the negative and positive samples.

Rapid detection of SARS-CoV-2 with CRISPR-Cas12a

Dan Xiong, Wenjun Dai, Jiaojiao Gong, Guande Li, Nansong Liu, Wei Wu, Jiaqiang Pan, Chen Chen, Yingzhen Jiao, Huina Deng, Junwei Ye, Xuanxuan Zhang, Huiling Huang, Qianyun Li, Liang Xue, Xiuming Zhang, Guanghui Tang