Development of an assay for the activity of Cas9 to improve the diagnostic ecosystem

This project began with the goal of developing novel CRISPR-based methods for SARS-CoV-2 variant detection. During development, we identified some limitations in existing CRISPR activity measurement techniques, which often required specialized equipment or complex processing steps. In response, we developed RepFluo, a new assay that employs melting curve analysis to monitor CRISPR-Cas9 DNA cleavage activity. The assay evolved through two iterations: an initial design using fluorescent DNA probes, and a simplified version utilizing SYBR Green dye that improved accessibility and reduced costs. We also developed a probe-based variation with potential multiplexing capabilities. We were able to successfully detect synthetic sequences from the human RPP30 gene and SARS-CoV-2 E gene, demonstrating clear discrimination between on-target and control samples through distinct melting temperature shifts. The assay operates on standard real-time PCR equipment, delivers results in under 15 minutes, and requires minimal sample processing. To support data analysis, we developed a specialized R package that provides a robust analytical framework. RepFluo represents an accessible approach to studying CRISPR activity while moving toward more sustainable molecular diagnostics.