(639f) Improving Sensitivity of CRISPR/Cas12-Based Biosensors Using Site-Specific Crosslinking of Cas12 Nuclease with Crrna

The CRISPR-based technologies hold great promise for molecular diagnostics. However, insufficient Cas nuclease-crRNA interaction limits the sensitivity of CRISPR systems. To address this issue, we genetically encoded latent bioreactive unnatural amino acids into Cas12 nuclease to site-specific crosslink with crRNA through proximity-enabled reactivity. The resulting site-specific covalent Cas12a-crRNA complex was leveraged to develop a fluorescent biosensor for nucleic acid detection. Notably, the detection sensitivity of the site-specific covalent Cas12a-crRNA system was substantially higher than that of the wild-type system. Combining with recombinase polymerase amplification (RPA) and in vitro transcription (IVT), the site-specific covalent Cas12a-crRNA system was successfully applied to detect Escherichia coli and Zika virus in clinic samples, achieving a high sensitivity and efficiency. Overall, our findings provide a promising avenue for improving CRISPR-based biosensors, with implications for broader applications in molecular diagnostics.