CRISPR/Cas12a-Based Aptasensor for Cas9 Protein Detection in Spiked Food Matrices

Abstract

Residual gene editors are a concern associated with the widespread application of CRISPR/Cas9 technology. Here, we developed a CRISPR/Cas12a-based fluorescent aptasensor for the qualitative and quantitative detection of Cas9 protein. Secondary structure prediction and molecular docking were combined to identify potential binding sites of the Cas9-specific aptamer. A fluorescence aptasensor was constructed based on competitive binding: in the presence of Cas9, the aptamer binds to the target and releases complementary DNA (cDNA), which activates the trans-cleavage activity of Cas12a. Cas12a subsequently cleaves a single-stranded DNA (ssDNA) reporter labeled with a fluorophore and a quencher, generating a fluorescence signal, expressed in relative fluorescence units (RFU), that increases with Cas9 concentration. The optimized conditions comprised a trans-cleavage reaction time of 20 min, a Cas12a:CRISPR RNA (crRNA) molar ratio of 1:1, a final aptamer concentration of 180 nM, and a combination of low- and high-salt buffers to balance molecular recognition and protein stability. The proposed assay enabled Cas9 protein detection in buffer and Cas9-spiked soybean milk and cereal matrices. These results represent a proof-of-concept evaluation in complex food matrices rather than validation of authentic residual Cas9 detection. Further studies using unspiked gene-edited samples with independently verified Cas9 residues and an orthogonal reference method are required.