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

Abstract

This study focuses on the core pain points of the current gene editing industry, specifically addressing the residual editor issue arising from the widespread application of CRISPR/Cas9 technology. 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.