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Construction of a CRISPR-Biolayer Interferometry Platform for Real-Time, Sensitive, and Specific DNA Detection
Shan-Peng Qiao1, Zhen-Ni Liu1, Hai-Chao Li1
1Department of Changchun Institute of Engineering Technology, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Science (CAS), 3333 Shengbei Street, Changchun, 130052, Jilin, P. R. China.
A novel CRISPR biosensor coupled with biolayer interferometry (CRISPR-BLI) enables rapid and sensitive DNA detection. This system utilizes modified deactivated CRISPR/Cas9 to identify specific DNA sequences with high accuracy.
Area of Science:
- Biotechnology
- Molecular Biology
- Biosensing
Background:
- Clustered regularly interspaced short palindromic repeats (CRISPR) technology offers high specificity for nucleic acid detection.
- Deactivated CRISPR/Cas9 (dCas9) can be engineered into sensitive biosensors.
Purpose of the Study:
- To develop a real-time, sensitive, and rapid digital system for double-stranded DNA detection.
- To leverage the specificity of CRISPR technology with the sensitivity of biolayer interferometry (BLI).
Main Methods:
- Modification of dCas9 with biotin to create a CRISPR biosensor.
- Immobilization of the CRISPR biosensor onto a biolayer for DNA capture.
- Coupling the biosensor with biolayer interferometry (BLI) to establish the CRISPR-BLI system.
- Detection of the ALP gene in Escherichia coli DH5α using the CRISPR-BLI system combined with PCR.
Main Results:
- The CRISPR-BLI system demonstrated real-time, sensitive, and rapid detection of double-stranded DNA.
- The system exhibited a linear detection range from 20 to 20,000 pg.
- A low detection limit of 1.34 pg was achieved for target DNA detection.
- The CRISPR biosensor maintained its DNA-recognition capability after preparation.
Conclusions:
- The CRISPR-BLI system is a promising approach for rapid and sensitive detection of target DNA analytes.
- This method offers a novel strategy for digital DNA detection with high specificity and sensitivity.
