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

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

doi:10.3389/fmolb.2023.1201347

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

Front Mol Biosci. 2023 Jun 14:10:1201347. doi: 10.3389/fmolb.2023.1201347. eCollection 2023.

Authors

Mustapha Aouida¹, Maryam Saifaldeen², Dana E Al-Ansari^{1

3}, Sara Taleb², Ali Ait Hssain^{1

4

5}, Dindial Ramotar¹

Affiliations

¹ College of Health and Life Sciences, Division of Biological and Biomedical Sciences, Hamad Bin Khalifa University, Education City, Qatar Foundation, Doha, Qatar.
² College of Health and Life Sciences, Division of Genomic and Precise Medicine, Hamad Bin Khalifa University, Education City, Qatar Foundation, Doha, Qatar.
³ National Heart and Lung Institute, Imperial College London, London, United Kingdom.
⁴ Medical ICU, Department of Medicine, Hamad Medical Corporation, Doha, Qatar.
⁵ Department of Medicine, Weill Cornell Medical College, Doha, Qatar.

Abstract

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.

Keywords: M gene; RT-RPA; SARS-CoV-2; dead Cas9; diagnostics; restriction enzymes.

Grants and funding

This project was funded by CHLS-HBKU internal funding and HBKU-innovation grant HBKU-INT-IC-SOCIF-05-01. We acknowledge the support received from Hamad Bin Khalifa University, Doha, Qatar, for providing funding. This project was funded through internal funding from CHLSHBKU and the HBKU Office of Innovation and Industrial Relations, Cycle 5 Social Innovation Funds grant. Last, the authors acknowledge the Qatar Foundation for supporting this work through the GSRA5-1-0602-18124 project and the Qatar National Research Funds—Qatar Research Leadership Program—ID: QRLP11-G-2003014.