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

Area of Science:

• Molecular Biology
• Biotechnology
• Virology

Background:

• Rapid, specific diagnostic kits are crucial for controlling global disease spread.
• CRISPR-based nucleic acid detection is a prominent method for diagnosing infections like COVID-19.

Purpose of the Study:

• To develop a novel, rapid, and highly specific CRISPR/Cas-based diagnostic tool for detecting SARS-CoV-2.
• To demonstrate the efficacy of the dead Cas9 Protects Restriction Enzyme Sites (dP-RES) approach for pathogen detection.

Main Methods:

• Utilized an *in vitro* dCas9-sgRNA system for targeted gene detection.
• Employed CRISPR/Cas multiplexing with dCas9-sgRNA-BbsI and dCas9-sgRNA-XbaI to inactivate restriction enzyme sites on the SARS-CoV-2 M gene.
• Tested the method on synthetic DNA, M gene expressed in human cells, and samples from infected individuals.

Main Results:

• Successfully demonstrated specific inactivation of restriction enzyme sites (BbsI and XbaI) on the SARS-CoV-2 M gene using dCas9-sgRNA complexes.
• Showed that the dP-RES approach can detect the M gene in human cells and clinical samples from SARS-CoV-2 infected individuals.
• Validated the specificity and robustness of the CRISPR-based detection method.

Conclusions:

• The dP-RES approach offers a rapid and highly specific method for detecting SARS-CoV-2.
• This CRISPR/Cas-based technique has significant potential for application as a diagnostic tool for various DNA/RNA pathogens.
• The study provides a proof of concept for a novel diagnostic strategy in infectious disease control.