Activation mechanism and integrated one-pot assay of chiral-like cRNA-enhanced CRISPR/Cas12a systems.

The 5'-repeat fragments released during pre-crRNA maturation are critical yet understudied components of the CRISPR/Cas12a system. Here, we demonstrate that engineered 5'-repeat fragments can potently activate Cas12a cleavage, with efficiency strongly dependent on the length of the 3'-spacer. Strikingly, complete truncation of the 3'-spacer generates a "chiral-like crRNA" conformation that induces a delayed-switch mode of Cas12a activation, fundamentally distinct from conventional mature crRNA. Leveraging this characteristic, we develop the delayed cleavage feature-mediated one-pot sensing strategy that resolves the long-standing challenge of incompatibility between Cas12a-based cleavage reaction and nucleic acid amplification, achieving a 1000-fold improvement in sensitivity over that of the conventional mature crRNA-mediated one-pot method. Furthermore, we integrate a cleavage-based one-pot assay with a portable temperature-controlled fluorescence imaging device to create an on-site diagnostic platform for high-throughput screening. Our study further advances the understanding of the crRNA-guided mechanism and facilitates the expansion of its applications in genome editing and molecular diagnostics.