CRISPR-Cas12a molecular diagnostics rely on target-activated collateral cleavage of single-stranded DNA (ssDNA) reporters to generate fluorescence signals, yet the mechanisms governing reporter substrate processing remain poorly understood. Here we show that Cas12a trans-cleavage involves both endonucleolytic cleavage and exonucleolytic trimming. We further demonstrate that terminal phosphate modification of ssDNA substrates accelerates substrate cleavage activity and modulates trans-cleavage kinetics, thereby enhancing fluorescence output. Guided by this mechanistic insight, we engineered fluorophore-quencher ssDNA reporters bearing terminal phosphate modifications and identified a 3'-phosphate-engineered reporter that enables faster signal generation and higher fluorescence intensity. Incorporation of the engineered reporter into the CRISPR-Cas12a system enabled the development of Phos-CRISPR (Phosphate-engineered DNA reporters boost CRISPR), a rapid, sensitive, and specific nucleic acid detection platform that achieves results within 20 min, approximately threefold faster than conventional CRISPR assays. Applied to herpes simplex virus 1 detection, the Phos-CRISPR combined with recombinase polymerase amplification achieved a limit of detection of 20 aM and accurately identified clinical swab samples. These findings provide a general framework for understanding Cas12a trans-cleavage activity and establish a mechanistically informed strategy for enhancing next-generation CRISPR diagnostics.
