Precise regulation of Cas12a activity is crucial for expanding its application in molecular diagnostics. However, existing split crRNA systems exhibit hardly any activation efficiency at low-abundance target and lack a well-defined regulated mechanism, representing a persistent bottleneck for practical application. This work proposes a DNA-guided spatially ordered assembly of split crRNA for activating CRISPR/Cas12a (DIRECTOR) strategy. This work combines artificial intelligence-driven AlphaFold3 structure prediction, computer-powered molecular dynamics simulations with fluorescence analysis to demonstrate that the 3' terminal extension of activator acts as a spatial director, utilizing DNA-guided spatially ordered assembly of split crRNA and stabilizing key Cas12a domains, thereby activating Cas12a. Conversely, the 5' terminal extension serves as a spatial misdirector, inhibiting Cas12a activation by destabilizing the protein structure and introducing the steric hindrance to shield the catalytic center. Furthermore, the structural and energy thresholds required for effective Cas12a activation were identified. Finally, utilizing the spatial director as an energy amplification element, DIRECTOR achieves a limit of detection as low as 42.1 fM for single-target miR-155 and dual-response detection of wide-scope nucleic acids. Owing to its direct activation strategy, DIRECTOR provides mechanistic insights for affordable and programmable CRISPR molecular diagnostics.
