5′-End Engineering of CRISPR/Cas12a Activators: A Versatile Platform for Multiple Biomarker Analysis and Clinical Cancer Tissue Identification

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.