The CRISPR/Cas12a system has emerged as a transformative tool in molecular diagnostics and biosensing, leveraging its high-efficiency DNA-targeting and unique-cleavage activity. However, its practical deployment is hindered by persistent challenges such as elevated background signals, constrained target versatility, and insufficient controllability. The activator strand (AS), serving as the molecular trigger for Cas12a activation, presents a promising engineering target to systematically enhance system performance. This review comprehensively summarizes recent advances in AS-driven regulation of the CRISPR/Cas12a system, focusing on four core engineering strategies: terminal modification engineering, split activator design, PAM (protospacer adjacent motif) engineering and regulation, and topological conformation engineering. By redesigning AS architecture, introducing allosteric control, and refining spatial assembly, these approaches significantly improve detection sensitivity, specificity, and versatility. AS engineering has effectively mitigated background interference, expanded target scope to include non-nucleic acid analytes, and enabled precise conditional activation of Cas12a. We further discuss current challenges and future directions, aiming to guide the development of next-generation CRISPR diagnostic systems with enhanced robustness, programmability, and adaptability for real-world applications. trans
