Conformationally Regulated CRISPR/Cas12a Activation Enabled by a Programmable DNA Dumbbell for Electrochemical SNP Genotyping

Single-nucleotide polymorphism (SNP) genotyping is crucial for genetic research and precision medicine, yet reliable discrimination of single-base variants in complex genomic backgrounds remains analytically challenging. Although CRISPR/Cas12a-based biosensing offers high sequence specificity, its intrinsic mismatch tolerance often leads to nonspecific activation by wild-type sequences, thereby compromising SNP fidelity. Herein, we report a highly sensitive electrochemical sensing platform based on a programmable DNA dumbbell (Dum) probe that functions as a conformational energy-barrier regulator of CRISPR/Cas12a activation. The closed-loop dumbbell architecture sterically shields the crRNA-activating sequence, establishing a high activation threshold that suppresses nonspecific Cas12a triggering. Only precise SNP hybridization induces a thermodynamically favorable conformational rearrangement, releasing the mediator and transitioning the CRISPR system from an inactive to an active state. To further enhance analytical sensitivity and reliability, nucleic acid-functionalized FeCo nanozymes were incorporated as catalytic signal transducers, enabling a self-validating dual-mode signal electrochemical readout through intrinsic metal redox and H₂O₂ electrocatalysis. The resulting platform achieved reliably discriminates mutation abundances down to 0.1%. Validation using soybean genomic DNA samples demonstrates the robustness and practical applicability of the proposed strategy. This work establishes a conformational energy-barrier-regulated CRISPR activation paradigm, providing a generalizable analytical framework for high-fidelity SNP genotyping in molecular breeding and clinical diagnostics.