All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus

Abstract

A recent outbreak of novel coronavirus (SARS-CoV-2), the causative agent of COVID-19, has spread rapidly all over the world. Human immunodeficiency virus (HIV) is another deadly virus and causes acquired immunodeficiency syndrome (AIDS). Rapid and early detection of these viruses will facilitate early intervention and reduce disease transmission risk. Here, we present an All-In-One Dual CRISPR-Cas12a (termed "AIOD-CRISPR") assay method for simple, rapid, ultrasensitive, one-pot, and visual detection of coronavirus SARS-CoV-2 and HIV virus. In our AIOD CRISPR assay, a pair of crRNAs was introduced to initiate dual CRISPR-Cas12a detection and improve detection sensitivity. The AIOD-CRISPR assay system was successfully utilized to detect nucleic acids (DNA and RNA) of SARS-CoV-2 and HIV with a sensitivity of few copies. Also, it was evaluated by detecting HIV-1 RNA extracted from human plasma samples, achieving a comparable sensitivity with real-time RT-PCR method. Thus, our method has a great potential for developing next-generation point-of-care molecular diagnostics.

Figure 1.

Design and working principle of the AIOD-CRISPR assay. (A) Schematic of the AIOD-CRISPR assay system. SSB, single-stranded DNA binding protein. (B) Development and evaluation of the AIOD-CRISPR assay system. The ssDNA-FQ reporter was labelled 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. HIV-1 p24 plasmid (1.2× 10⁵ copies), 8 μM of ssDNA-FQ reporters, and 0.64 μM EnGen® Lba Cas12a (Cpf1) were used. (i) Eight reaction systems with various components and their endpoint images after 40 min incubation. (ii) Denaturing PAGE analysis of the AIOD-CRISPR products. (iii) Real-time fluorescence detection of the AIOD-CRISPR assay for eight reaction systems with various components.

Figure 2.

Comparison of the all-in-one CRISPR-Cas12a assay using dual crRNAs or single crRNA. (A) The pUCIDT-AMP plasmid containing 300 bp HIV-1 p24 gene cDNA (p24 plasmid) and the sequences of its primers and crRNAs (B) Real-time fluorescence detection of the all-in-one CRISPR-Cas12a assay using dual crRNAs (crRNA1+crRNA2) or single crRNA (crRNA1/crRNA2). 2*crRNA1/2*crRNA2 means doubling its amount. P, the positive reaction with 1.2× 10³ copies of HIV-1 p24 plasmids. (C) The CRISPR-Cas12a assays with dual crRNAs (crRNA1+crRNA2) or crRNA2 for the detection of 1.2 copies of HIV-1 p24 plasmids (P). Three replicates ran for each reaction with the plasmid. NTC, non-template control reaction. Each reaction contained 2 μM ssDNA-FQ. Fluorescence images were taken after 40 min incubation.

Figure 3.

HIV-1 p24 plasmid (DNA) detection by the AIOD-CRISPR assay. (A) Real-time detection and endpoint fluorescence/visual detection. (B) Denaturing PAGE analysis of the AIOD-CRISPR products. (C) Endpoint fluorescence comparison after 1 min incubation at 37 °C. NTC, non-template control reaction. Three replicates ran for each reaction or test. Error bars represent the standard deviations at three replicates (n = 3). Unpaired two-tailed t-test was used to analyse the difference from NTC. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant.

Figure 4.. HIV-1 RNA detection by the RT-AIOD-CRISPR assay.

(A) Sensitivity of real-time and endpoint fluorescence/visual detections. Three replicates were conducted for each test. NTC, non-template control reaction. Error bars represent the standard deviations at three replicates (n = 3). Unpaired two-tailed t-test was used to analyse the difference from NTC. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant. (B) Real-time and visual AIOD-CRISPR assays for the detection of HIV-1 RNA extracted from human plasma samples. (C) Real-time RT-PCR assay to detect HIV-1 RNA extracted from human plasma samples. Negative, HIV-1 negative plasma samples.

Figure 5.. SARS-CoV-2 N DNA detection by the AIOD-CRISPR assay.

(A) The pUCIDT-AMP plasmid containing 316 bp SARS-CoV-2 N gene cDNA (N plasmid) and the primers and crRNAs. (B) Real-time AIOD-CRISPR detection with various copies of SARS-CoV-2 N DNA. (C) Specificity assay of the AIOD-CRISPR assay on SARS-CoV-2 N detection. Tube images were taken after 40 min incubation.

Figure 6.. SARS-CoV-2 RNA detection by the RT-AIOD-CRISPR assay.

(A) Protocol and PCR primers for preparing the SARS-CoV-2 N RNA sequences. (B) Sanger sequencing of the AIOD-CRISPR detection region in the prepared SARS-CoV-2 N RNA. (C) Sensitivity of real-time and endpoint fluorescence/visual AIOD-CRISPR detections. NTC, non-template control reaction. Three replicates were conducted for each test. Error bars represent the standard deviations at three replicates (n = 3). Unpaired two-tailed t-test was used to analyse the difference from NTC. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant.