biostudies-literatureZhu ZFundamental Research Funds for the Central UniversitiesNational Natural Science Foundation of ChinaHuazhong University of Science and Technologygkaf311https://www.ebi.ac.uk/biostudies/studies/S-EPMC12014286biostudies-literatureUnknown53(8)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.Nucleic acids researchExploring the effect of activator topology on CRISPR-Cas12a trans-cleavage activity.PMC120142862024JYCXJJ0108217237222474045Li XZhu ZWu TDing LfalseExploring the effect of activator topology on CRISPR-Cas12a trans-cleavage activity.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.2025-01-01T00:00:00Z2025 Apr2025-07-02T03:05:02.769Z2025-07-02T03:05:02.769ZS-EPMC120142864026370710.1093/nar/gkaf311