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Published on: December 23, 2022
Low-Background CRISPR/Cas12a Sensors for Versatile Live-Cell Biosensing.
Qing-Nan Li1, Dong-Xia Wang1, Gui-Mei Han1
1State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Centre for Analytical Sciences, College of Chemistry, Nankai University, Tianjin 300071, P. R. China.
Researchers developed a novel CRISPR/Cas12a biosensor using a "RESET" activator strand to overcome high background issues. This innovation enables sensitive detection in live cells and point-of-care applications.
Area of Science:
- Biotechnology
- Molecular Biology
- Biosensing
Background:
- CRISPR/Cas12a's trans-cleavage activity is vital for biosensing.
- Existing "on-off-on" CRISPR/Cas12a biosensors face high background noise, limiting intracellular use.
Purpose of the Study:
- To overcome high background in CRISPR/Cas12a biosensors.
- To enable sensitive detection and intracellular applications.
- To enhance biosensor flexibility for diverse targets.
Main Methods:
- Designed a CRISPR/Cas12a activator strand (AS) utilizing the "RESET" effect for controllable activation.
- Developed strategies including strand displacement, cleavage, and aptamer-substrate interactions to reactivate inhibited Cas12a activity.
- Integrated biosensors with lateral flow assay (LFA) for point-of-care testing.
Main Results:
- Achieved low background noise by inhibiting AS activation.
- Demonstrated sensitive detection of nucleic acid (miR-21, 0.96 pM), ATP (8.6 μM), and hOGG1 (8.3 × 10-5 U/mL).
- Successfully performed intracellular imaging of biomolecules and integrated with LFA.
Conclusions:
- The "RESET" effect significantly improves CRISPR/Cas12a biosensor performance and flexibility.
- Developed biosensors are suitable for live-cell analysis and point-of-care diagnostics.
- This approach broadens the applicability of CRISPR/Cas12a in various sensing scenarios.
