A CRISPR-based approach using dead Cas9-sgRNA to detect SARS-CoV-2

• Published in: Frontiers in molecular biosciences Vol. 10; p. 1201347
• Main Authors: Aouida, Mustapha, Saifaldeen, Maryam, Al-Ansari, Dana E., Taleb, Sara, Hssain, Ali Ait, Ramotar, Dindial
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 14.06.2023
• Subjects: dead Cas9; diagnostics; M gene; Molecular Biosciences; restriction enzymes; RT-RPA; SARS-CoV-2; M gene; SARS-CoV-2; diagnostics; RT-RPA; dead Cas9; restriction enzymes
• ISSN: 2296-889X; 2296-889X
• DOI: 10.3389/fmolb.2023.1201347

Rapid, highly specific, and robust diagnostic kits to detect viruses and pathogens are needed to control disease spread and transmission globally. Of the many different methods proposed to diagnose COVID-19 infection, CRISPR-based detection of nucleic acids tests are among the most prominent. Here, we describe a new way of using CRISPR/Cas systems as a rapid and highly specific tool to detect the SARS-CoV-2 virus using the in vitro dCas9-sgRNA-based technique. As a proof of concept, we used a synthetic DNA of the M gene, one of the SARS-CoV-2 virus genes, and demonstrated that we can specifically inactivate unique restriction enzyme sites on this gene using CRISPR/Cas multiplexing of dCas9-sgRNA- BbsI and dCas9-sgRNA- XbaI . These complexes recognize and bind to the target sequence spanning the BbsI and XbaI restriction enzyme sites, respectively, and protect the M gene from digestion by BbsI and/or XbaI . We further demonstrated that this approach can be used to detect the M gene when expressed in human cells and from individuals infected with SARS-CoV-2. We refer to this approach as dead Cas9 Protects Restriction Enzyme Sites, and believe that it has the potential to be applied as a diagnostic tool for many DNA/RNA pathogens.