Mismatch-Engineered CRISPR Transistors for Ultra-Low-Frequency Single-Nucleotide Variant Detection

Abstract Accurate detection of low-frequency single-nucleotide variants (SNVs) in a high wild-type (WT) background remains challenging. Here, we report a mismatch-engineered CRISPR transistor (MECT) for rapid, amplification-free detection of mutant RNA. MECT integrates a Cas13a–crRNA recognition interface with a graphene field-effect transistor and employs a spaced mismatch crRNA design centered on the mutation site in a nonseed region. It leverages the synergistic effect between natural and engineered mismatches at the mutation site to weaken WT binding while preserving mutant recognition, thereby amplifying the difference in response between mutants and the WT. Using BRAF V600E RNA as a target, MECT generates a mutant response approximately 11.58 times stronger than the WT background, with a detection limit of 3 × 10–18 mol L–1. MECT can also detect mutants as low as 0.0001% against a WT background. A portable MECT-based prototype is validated using cell-derived RNA and clinical samples, supporting decentralized analysis of low-frequency cancer mutations.