dPAFS: A nuclease-dead PfAgo-mediated fluorescence sensing platform for genotyping genome-edited rice

Pyrococcus furiosus Argonaute (PfAgo)-mediated nucleic acid detection is a powerful tool for molecular diagnostics, offering ultrahigh sensitivity and single-nucleotide specificity. However, existing PfAgo-based methods require multi-step cleavage processes and the use of molecular beacons, which are cumbersome and costly. Herein, we developed a novel fluorescence sensing platform (dPAFS) based on a nuclease-dead PfAgo mutant (dPfAgo) to simplify PfAgo-mediated detection system significantly. dPfAgo mutants were obtained by site-directed mutagenesis of the key catalytic site residues D628 and D558, and their functionality was confirmed through activity assay. In this sensing system, the gDNA was modified with the quenched group of black hole quencher-1 (BHQ1). Target DNA was amplified with carboxyfluorescein (FAM)-labeled primers and then precisely bound with gDNA-dPfAgo. The formed gDNA-dPfAgo-tDNA ternary complex brought the FAM donor and BHQ1 acceptor into close proximity, inducing fluorescence quenching through fluorescence resonance energy transfer. In a proof-of-concept study, dPAFS successfully genotyped genome-edited rice variants, achieving a limit of detection of 0.1 % for edited-type variant and distinguishing variants of genome-edited rice with single-nucleotide specificity. The dPAFS platform eliminates the need for molecular beacons, offering a simple, cost-efficient, and robust assay for programmable enzyme-mediated molecular diagnoses.