Strand Displacement-Driven CRISPR/Cas12a Framework Enables Precise Identification of Low-Abundance Point Mutations

Accurate identification of low-abundance point mutations is a crucial technical challenge in clinical diagnosis, particularly within high wild-type genomic backgrounds. We established an engineered CRISPR/Cas12a point mutation detection strategy (E-CRISPR) for low-abundance point mutations detection. By integrating the precise regulatory mechanism of strand displacement reaction (SDR) with the local high-concentration probe microenvironment created by tetrahedral DNA frameworks (TDFs), it has achieved simultaneous improvement in single-nucleotide resolution and ultrasensitive detection performance. This strategy was validated using an mitochondrial m.11778 G>A mutation associated with Leber’s hereditary optic neuropathy (LHON) and achieved a detection limit of 0.1% mutant allele frequency under isothermal conditions within 20 minutes. This approach represents a clinically versatile mutation diagnosis strategy with high sensitivity and specificity, which provides an innovative solution to breakthrough the existing technical bottlenecks.