Frontiers in Molecular Biosciences (Jun 2023)

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

  • Mustapha Aouida,
  • Maryam Saifaldeen,
  • Dana E. Al-Ansari,
  • Dana E. Al-Ansari,
  • Sara Taleb,
  • Ali Ait Hssain,
  • Ali Ait Hssain,
  • Ali Ait Hssain,
  • Dindial Ramotar

DOI
https://doi.org/10.3389/fmolb.2023.1201347
Journal volume & issue
Vol. 10

Abstract

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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.

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