CRISPR/Cas12a-Based Aptasensor for Cas9 Protein Detection in Spiked Food Matrices
- 1College of Life Science and Technology, Beijing University of Chemical Technology, Chaoyang District, Beijing 100029, China
- 2Quality and Safety Institute of Agricultural Products, Heilongjiang Academy of Agricultural Sciences/Key Laboratory of Quality and Safety of Cereals and Their Products, Harbin 150086, China
- 3Hangzhou Institute of Medicine (HIM), The Chinese Academy of Sciences, Hangzhou 310022, China
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.
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BibTeX
@article{wang2026crisprcas12abased,
title = {{CRISPR/Cas12a-Based Aptasensor for Cas9 Protein Detection in Spiked Food Matrices}},
author = {Zizhao Wang and Jing Wang and Ling Li and Guofeng Chen and Hongtao Wen and Baohai Liu and Yiyang Dong},
journal = {Molecules},
year = {2026},
month = oct,
doi = {10.3390/molecules31203607},
url = {https://www.mdpi.com/1420-3049/31/20/3607},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - CRISPR/Cas12a-Based Aptasensor for Cas9 Protein Detection in Spiked Food Matrices
AU - Zizhao Wang
AU - Jing Wang
AU - Ling Li
AU - Guofeng Chen
AU - Hongtao Wen
AU - Baohai Liu
AU - Yiyang Dong
PY - 2026
DA - 2026/10/01
JO - Molecules
DO - 10.3390/molecules31203607
UR - https://www.mdpi.com/1420-3049/31/20/3607
AB - 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.
SN - 1420-3049
ER -