Abstract

Amplification‐free Cas12a diagnostics with split crRNA enable rapid and programmable target recognition, yet insufficient understanding of DNA activator architecture prevents predictable control over trans ‐cleavage activity and sensitivity. Here we systematically map over 200 split DNA activator configurations by introducing nicks at every position across both strands. The mapping reveals that target strand nicks suppress activity with position‐dependent severity, while non‐target strand nicks enhance activity. Guided by these rules, we engineer an optimized split activator pair that achieves attomolar microRNA detection (LOD: 112 aM), ∼480‐fold higher sensitivity than intact activators. The enhanced sensitivity supports multiplexed live‐cell profiling of five miRNAs for machine learning‐based cancer cell stratification, and is further generalized to non‐nucleic acid targets, including APE1 enzyme (0.0073 U/L) and HClO (2.37 pM) through position‐informed cleavable sites. This work provides a generalizable methodology for engineering CRISPR‐Cas12a performance across diagnostic and biosensing applications.