Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay

Abstract

The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Rapid and early detection of SARS-CoV-2 facilitates early intervention and prevents the disease spread. Here, we present an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for one-pot, ultrasensitive, and visual SARS-CoV-2 detection. By targeting SARS-CoV-2's nucleoprotein gene, two CRISPR RNAs without protospacer adjacent motif (PAM) site limitation are introduced to develop the AIOD-CRISPR assay and detect the nucleic acids with a sensitivity of few copies. We validate the assay by using COVID-19 clinical swab samples and obtain consistent results with RT-PCR assay. Furthermore, a low-cost hand warmer (~$0.3) is used as an incubator of the AIOD-CRISPR assay to detect clinical samples within 20 min, enabling an instrument-free, visual SARS-CoV-2 detection at the point of care. Thus, our method has the significant potential to provide a rapid, sensitive, one-pot point-of-care assay for SARS-CoV-2.

Fig. 1. Design and working principle of the AIOD-CRISPR assay.

a Schematic of the AIOD-CRISPR assay system. SSB is single-stranded DNA binding protein. The four sites in the target sequence are labeled as F, T1, T2, and R, respectively. The letter c represents the corresponding complementary site. For example, F and Fc sites are complementary. The short horizontal lines with the same colors denote the same sites and their arrows represent the direction of 5′–3′. b Evaluation of eight AIOD-CRISPR reactions (R) with various components through endpoint imaging after 40-min incubation, denaturing polyacrylamide gel electrophoresis (PAGE) analysis of the single-stranded fluorescent reporter (ssDNA-FQ), and real-time fluorescence detection. The ssDNA-FQ was labeled by 5′ 6-FAM (Fluorescein) fluorophore and 3′ Iowa Black FQ quencher. Recombinase polymerase amplification (RPA) mix from TwistAmp Liquid Basic kit was composed of 1× Reaction Buffer, 1× Basic E-mix, 1× Core Reaction Buffer, 14 mM MgOAc, 320 nM each of primers, and 1.2 mM dNTPs. Dual crRNAs contained 0.64 μM each of crRNAs specific to the SARS-CoV-2 N gene sequence. A plasmid containing the SARS-CoV-2 N gene sequence (3 × 10³ copies), 8 μM of ssDNA-FQ reporters, and 1.28 μM EnGen Lba Cas12a (Cpf1) were used. Each experiment was repeated three times with similar results.

Fig. 2. Primer design and the sensitivity of AIOD-CRISPR for the detection of the tenfold serial dilution of plasmid DNA containing SARS-CoV-2 N gene sequence (N plasmid).

a SARS-CoV-2 genome map with the detailed sequence information of the designed primers and crRNAs targeting the N gene sequence. For comparison purpose, the designed crRNA1 and crRNA2 were not limited by the PAM site (5′-TTTG-3′), while the crRNA3 was limited by the PAM site. b Real-time/endpoint AIOD-CRISPR assay using dual crRNAs (crRNA1&2) without PAM site limitation. Source data are provided as a Source Data file. c Real-time/endpoint AIOD-CRISPR assay using single crRNA (crRNA3) with PAM site limitation. Four replicates were run (n = 4). Source data are provided as a Source Data file. The horizontal dashed line indicates the cutoff fluorescence that was defined by the average intensity of NTC plus three times of the standard deviation. NTC, non-template control reaction. Error bars represent the means ± standard deviation (s.d.) from replicates. The unpaired two-tailed t-test was used to analyze the statistical significance. Source data is available for b, c.

Fig. 3. Specificity of AIOD-CRISPR and sensitivity of RT-AIOD-CRISPR for detection of synthetic SARS-CoV-2 N RNA sequences.

a Specificity of real-time/endpoint AIOD-CRISPR assay for SARS-CoV-2 N detection. Three replicates were run (n = 3). Source data are provided as a Source Data file. b Real-time/endpoint RT-AIOD-CRISPR detection of the tenfold serial dilution of synthetic SARS-CoV-2 N RNA sequences. Four replicates were run (n = 4). Source data are provided as a Source Data file. The horizontal dashed line indicates the cutoff fluorescence that was defined by the average intensity of NTC plus three times of the standard deviation. NTC, non-template control reaction. Error bars represent the means ± s.d. from replicates. The unpaired two-tailed t-test was used to analyze the statistical significance. Source data is available for a, b.

Fig. 4. Detection of SARS-CoV-2 in clinical swab samples by RT-AIOD-CRISPR assay.

a Real-time RT-AIOD-CRISPR detection. b Endpoint fluorescence/visual detection after 40 min incubation. PC, positive controls with 4.6 × 10⁴ copies of synthetic SARS-CoV-2 N RNA. S1-S28, clinical samples 1-28. NC, SARS-CoV-2-negative control reaction. NTC non-template control reaction.

Fig. 5. Instrument-free COVID-19 diagnostics by RT-AIOD-CRISPR assay.

a Workflow of the instrument-free POC diagnostics. b Visual detection after 20, 40, and 60-min incubation on the hand warmer bag. c Analysis of the green value for the fluorescence image (20-min incubation) using the ImageJ software. The horizontal dashed line indicates the cutoff fluorescence that was defined by the average intensity of NTC plus three times of the standard deviation. Source data are provided as a Source Data file. NC, the SARS-CoV-2-negative sample. Each measuring was run with three replicates (n = 3). Error bars represent the means ± s.d. from replicates. The unpaired two-tailed t-test was used to analyze the statistical significance. Source data is available for c.