Detection of Infectious Viruses Using CRISPR-Cas12-Based Assay

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• The COVID-19 pandemic caused by SARS-CoV-2 significantly impacted global health and economies.
• CRISPR-Cas-based assays are emerging as powerful tools for pathogen detection due to their sensitivity, specificity, and ease of use.

Purpose of the Study:

• To develop and validate a CRISPR-Cas12-based assay for the rapid and sensitive detection of SARS-CoV-2.
• To assess the assay's performance using clinical samples and its adaptability for other viral pathogens.

Main Methods:

• Development of a CRISPR-Cas12a assay combined with isothermal reverse transcription recombinase polymerase amplification (RT-RPA) for target amplification.
• Utilized CRISPR-Cas12a/guide RNA complexes and collateral cleavage of fluorophore-tagged probes for detection via fluorescence or lateral flow strips.
• Validated the assay using nasal swabs and sputum from COVID-19 patients.

Main Results:

• The developed CRISPR-Cas12 assay achieved sensitive detection of SARS-CoV-2 down to 10 copies per sample.
• The assay demonstrated reliability when tested with clinical specimens from COVID-19 patients.
• The assay's core scheme was successfully adapted for the detection of Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV).

Conclusions:

• The CRISPR-Cas12-based assay provides a sensitive, specific, and versatile platform for SARS-CoV-2 detection.
• This assay holds significant potential for development into a point-of-care testing (POCT) tool for various infectious viral diseases.
• The adaptability of the assay highlights its broad applicability in infectious disease diagnostics.