<b>A Qualitative and Quantitative Detection Method for Gene Editor Residues Based on Cutting-Edge Biosensing Technology</b>

A novel, highly sensitive method for the qualitative and quantitative detection of residual Cas9 has been developed, which integrates CRISPR/Cas12a with aptamer-based recognition.By combining secondary structure prediction and tertiary structure molecular docking, the key binding sites of the Cas9-specific aptamer were validated.

A fluorescence aptasensor based on a competitive binding mechanism was then constructed: in the presence of residual Cas9, the aptamer preferentially binds to the target, releasing complementary DNA (cDNA) that activates the trans-cleavage activity of Cas12a. This activity cleaves a single-stranded DNA (ssDNA) probe dual-labeled with a fluorophore and a quencher, generating a fluorescence signal (relative fluorescence units, RFU) that is positively correlated with the Cas9 concentration.Systematic optimization experiments determined the optimal detection conditions: a 20-minute trans-cleavage duration, a 1:1 molar ratio of Cas12a to CRISPR RNA (crRNA), a final aptamer concentration of 180 nM, and a combination of high- and low-salt buffers to balance molecular recognition specificity and protein stability.In conclusion, this study provides a low-cost, highly sensitive detection strategy, offering a novel technical pathway for the safety regulation of gene-edited products.</p>