Exploring the effect of activator topology on CRISPR–Cas12a trans-cleavage activity

Abstract The CRISPR–Cas12a system is widely used in nucleic acid detection and biosensing due to its high sensitivity, selectivity, and simple design. However, traditional CRISPR–Cas12a sensors, which rely on linear activators, face challenges such as limited operability and low stability. This study explored the impact of three different activator topologies—linear, planar, and steric—on the trans-cleavage activity of Cas12a. We developed a Cas12a-based switch using a planar activator, which demonstrated superior operability and maintained higher activity compared to linear activators. Using this planar activator, we achieved highly sensitive detection of hypochlorous acid, with a detection limit as low as 88 nM, outperforming chemical probe-based methods. The introduction of topological activators will open new avenues for the development of CRISPR–Cas12a-based biosensors, offering broad potential for diverse applications.