Steric regulation of CRISPR/Cas12a trans -cleavage kinetics via split-activator extensions

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a holds substantial promise for molecular diagnostics, yet its rapid and uncontrolled activation often results in background leakage and disrupts the coordination of upstream reaction modules. Here, we established a steric-regulation framework that enables predictable tuning of Cas12a trans-cleavage kinetics through rationally engineered extensions on split activators. Systematic analysis of extension orientation, length, and hybridization state revealed quantitative and direction-dependent rules governing steric control of activator assembly and Cas12a activation. Guided by these insights, we integrated the sterically regulated split activator into an entropy-driven DNA circuit to construct a fully one-pot cascaded detection system. The engineered steric barriers effectively suppressed premature activation and established precise kinetic matching between the DNA circuit and Cas12a. The resulting platform achieved a detection limit of 1.24 pM for microRNA-21 and demonstrated high fidelity. This work defines a predictable steric-gating mechanism for Cas12a activation and delivers a nucleic-acid-only regulatory module that can be incorporated into diverse CRISPR architectures, supporting the development of robust, leakage-resistant one-pot diagnostic systems.