Detection of Infectious Viruses Using CRISPR-Cas12-Based Assay

Abstract

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease-19 (COVID-19), has severely influenced public health and economics. For the detection of SARS-CoV-2, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)-based assays have been emerged because of their simplicity, sensitivity, specificity, and wide applicability. Herein, we have developed a CRISPR-Cas12-based assay for the detection of SARS-CoV-2. In the assay, the target amplicons are produced by isothermal reverse transcription recombinase polymerase amplification (RT-RPA) and recognized by a CRISPR-Cas12a/guide RNA (gRNA) complex that is coupled with the collateral cleavage activity of fluorophore-tagged probes, allowing either a fluorescent measurement or naked-eye detection on a lateral flow paper strip. This assay enables the sensitive detection of SARS-CoV-2 at a low concentration of 10 copies per sample. Moreover, the reliability of the method is verified by using nasal swabs and sputum of COVID-19 patients. We also proved that the current assay can be applied to other viruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), with no major changes to the basic scheme of testing. It is anticipated that the CRISPR-Cas12-based assay has the potential to serve as a point-of-care testing (POCT) tool for a wide range of infectious viruses.

Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities

“…For instance, two different groups [ 27 , 28 ] reported an LOD of 10 copies/µL using CRISPR/Cas12a, LAMP, and commercial Milenia HybriDetect dipsticks for colorimetric detection and a laboratory-centered fluorophotometer. Similarly, by substituting LAMP with RPA, Talwar et al [ 2 ] and Gong et al [ 31 ] also achieved LODs of 10 and 5 copies/reaction, respectively. In the HybriDetect dipstick, a test line is immobilized with an anti-goat antibody that captures AuNPs with goat anti-FITC antibody, whereas the control line is coated with a streptavidin that captures the FITC-biotinylated reporter.…”

“…As such, the signal at the test line could solely be obtained without switching the test and control lines to prevent intuitive misinterpretation. More importantly, since CRISPR/Cas12a machinery relies on three main elements (Cas12a, crRNA, and the reporter) to achieve nucleic acid detection, we were able to design and transduce the signal directly from the CRISPR/Cas12a machinery without using an auxiliary carrier, such as biotin, streptavidin, or digoxin, as observed in several CRISPR/Cas12a-based LFA [ 2 , 25 , 27 , 28 , 29 , 30 , 31 ] ( Table 1 ). The concept of CRICOLAP LFS can also serve as a state of the art to harness the trans-cleavage activity, which we believe can increase sensitivity and specificity, because the signal reporter of the CRISPR/Cas machinery is directly captured by a signal amplifier (capture probe) at the test line.…”

“…The novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a threat to global public health with a widespread infection of 236 million cases and 4.8 million deaths as of 5 October 2021 [ 1 ]. The SARS-CoV-2 outbreak has an evolutionary genetic connection with the coronaviruses that caused SARS-CoV and MERS-CoV pneumonia in Guangdong Province in 2003 and in the Middle East in 2012, respectively [ 2 ].…”

Development of a Rapid and Efficient RPA-CRISPR/Cas12a Assay for Mycoplasma pneumoniae Detection

“…Additionally, cleavage results of CRISPR/Cas12a system can be visualized using lateral flow biosensor (LFB) or fluorescence reader by introducing ssDNA reporter labeled with fluorophore and biotin or alternatively with fluorophore and quencher into an in vitro reaction (Xiong et al, 2020;Zhu et al, 2021). Now, CRISPR/Cas12a detection integrated with RPA has been successfully applied in rapid and accurate detection of various pathogens including viral pathogens (such as SARS-CoV-2, human metapneumovirus, human papillomavirus and African swine fever; Bai et al, 2019;Yin et al, 2020;Qian et al, 2021;Sun et al, 2021;Talwar et al, 2021), fungal pathogens (for example Elsinoë fawcettii; Shin et al, 2021), Bacterial pathogens (such as Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus, Salmonella sp., Xanthomonas arboricola and Pseudomonas aeruginosa; Cai et al, 2021;Liu et al, 2021;Luo et al, 2021). In this study, RPA coupled with CRISPR/Cas12a (RPA-CRISPR/Cas12a) has been developed for MP detection.…”

Recombinase Polymerase Amplification for Rapid Detection of Zoonotic Pathogens

“…Cas system has recently been used to sensitively detect nucleic acids, and Numerous CRISPR-Cas-RPA detection systems have been developed[125][126][127][128]. Most of these studies are based on combination of CRISPR/Cas12a and RT-RPA, with introduction of fluorescence probe for fluorescence readout or gold nanoparticles (AuNPs) for colorimetric readout[31,122,126,127,[129][130][131][132].For example, the technique of the CRISPR-Cas12-based assay combine with the DNA-modified gold nanoparticles (AuNPs) has been developed, making the detection limit to 1 copy of viral genome sequence per test.But the detection time has been increased to 50 minutes, of which 30 minutes were used for colorimetric readings[122,126,127]. Two separate scientific teams combined CRISPR/Cas9, LF assay, and RT-RPA technology as a platform for visual detection of SARS-CoV-2, providing an accurate and convenient pathway for diagnosis of COVID-19 or other infectious diseases in resource-limited regions[128,133].…”