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Updated: Sep 11, 2025
Author Spotlight: Engineering Molecular Tools for Disease Detection and Imaging
Published on: December 8, 2023
5'-End Engineering of CRISPR/Cas12a Activators: A Versatile Platform for Multiple Biomarker Analysis and Clinical
Jia-Yi Shi1, Zi-Wen Li1, Zhi-Li Yao2
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.
Abstract:
The CRISPR/Cas12a system has emerged as a powerful tool for biosensing due to its unique trans-cleavage activity. However, the fundamental mechanisms governing its activation remain inadequately understood, limiting the design flexibility and application scope of CRISPR/Cas12a-based biosensors. In this study, we investigated the activation behavior of CRISPR/Cas12a, focusing on the 5'-end engineering of the activator strand. We discovered that the activation of CRISPR/Cas12a can be significantly suppressed by incorporating a rigid intramolecular hairpin or intermolecular duplex at the 5'-end of the activator strand designed using our discovered RESET effect. Leveraging this finding, we developed a series of CRISPR/Cas12a-based biosensors capable of sensitive and selective detection, as well as live-cell imaging, for various biomarkers including microRNAs, biological small molecules, enzymes, and reactive oxygen species. Notably, the biosensor designed for miR-210, a biomarker for renal cell carcinoma (RCC), demonstrated exceptional performance in distinguishing between clinical RCC tissues and adjacent healthy tissues, highlighting its potential for cancer diagnosis, prognosis, and intraoperative decision-making. This study not only deepens the understanding of CRISPR/Cas12a activation mechanisms but also provides a versatile platform for developing advanced biosensors in molecular diagnostics and therapeutic monitoring.
Insights
Researchers engineered the CRISPR/Cas12a system by modifying the activator strand, creating a novel RESET effect. This breakthrough enhances CRISPR/Cas12a biosensor sensitivity and selectivity for diverse biomarker detection and diagnostics.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- CRISPR/Cas12a is a potent biosensing tool leveraging trans-cleavage activity.
- Limited understanding of CRISPR/Cas12a activation mechanisms restricts biosensor design and application.
- The 5'-end of the activator strand is crucial for CRISPR/Cas12a function.
Purpose of the Study:
- To elucidate the activation mechanisms of CRISPR/Cas12a.
- To investigate the impact of 5'-end modifications on activator strand function.
- To develop advanced CRISPR/Cas12a-based biosensors with enhanced capabilities.
Main Methods:
- Investigated CRISPR/Cas12a activation by engineering the 5'-end of the activator strand.
- Utilized a novel RESET effect involving hairpin or duplex formation.
- Developed and validated CRISPR/Cas12a biosensors for various biomarkers.
Main Results:
- Discovered that 5'-end engineering via the RESET effect can suppress CRISPR/Cas12a activation.
- Developed highly sensitive and selective CRISPR/Cas12a biosensors for microRNAs, small molecules, enzymes, and reactive oxygen species.
- Demonstrated successful application in live-cell imaging and distinguishing cancer tissues (miR-210 for RCC).
Conclusions:
- The study provides a deeper understanding of CRISPR/Cas12a activation mechanisms.
- The RESET effect offers a versatile strategy for designing robust CRISPR/Cas12a biosensors.
- Developed biosensors show significant potential for molecular diagnostics, cancer detection, and therapeutic monitoring.
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