Phosphate‐Engineered DNA Reporters Boost CRISPR‐Cas12a Trans ‐Cleavage for Rapid and Sensitive Nucleic Acid Detection

ABSTRACT

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 ( Phos phate‐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.