PAM-Deleted Hairpin Gating Enables Low-Background One-Pot DNA Circuit-Cas12a Coupling for SERRS Detection of Genetically Modified Crops

Abstract CRISPR/Cas-based nucleic acid detection often relies on polymerase-driven preamplification and multistep workflows. One-pot detection is always a desired goal. However, one-pot integration is hindered by background activation and mismatched reaction requirements. Inadvertent protospacer-adjacent motif (PAM) presentation within hairpin intermediates is a key source of premature Cas12a activation, leading to a non-specific background. Here, we develop a preamplification-free assay that addresses this one-pot coupling problem by integrating an enzyme-free catalytic hairpin assembly and hybridization chain reaction (CHA/HCR) circuit with CRISPR/Cas12a under a single isothermal condition. A PAM-deleted hairpin gating strategy is employed to reduce premature Cas12a activation and improve one-pot compatibility between the CHA/HCR circuit and Cas12a, thereby suppressing non-specific background. The CHA/HCR reaction assembles repetitive crRNA-addressable motifs to rapidly activate Cas12a, and then the activated Cas12a cleaves the G-rich ssDNA above the magnetic-plasmonic particle (Fe3O4/AgNPs/ssDNA), preventing G-quadruplex formation and abolishing methylene blue loading, thereby yielding a turn-off surface-enhanced resonance Raman scattering (SERRS) output. The assay is completed within 90 min and has a limit of detection (LOD) of 4.65 fM (2.8 × 103 copies/μL). It further enables the accurate classification of genetically modified maize samples, including seed-derived samples and leaf-extracted RNA, with high concordance with qPCR results. This nucleic acid detection platform streamlines the assay workflow and provides a broadly applicable route for on-site sensing via one-pot isothermal CHA/HCR−CRISPR/Cas12a coupling with SERRS readout.