Exploiting Asymmetric Mismatch Sensitivity of Cas13a for Interference-Tolerant Single-CpG Methylation Analysis

Abstract Accurate single-CpG methylation analysis in dense regions is critical for cancer diagnostics but challenged by adjacent CpG interference. Here, we developed EPICATCH, an interference-tolerant ratiometric Cas13a biosensor to overcome this limitation. Exploiting LbuCas13a′s asymmetric mismatch sensitivity, we established a dual-zone recognition framework. Positioning the target CpG in the strictly sensitive 5′-zone guarantees single-base resolution, while placing adjacent CpGs in the tolerant 3′-zone masks epigenetic noise, achieving a 7-bp spatial resolution for the tested MYO1G construct. Integrated with recombinase polymerase amplification, ssDNA generation, and a dual-channel ratiometric readout, EPICATCH eliminates sample concentration biases, achieving a 100 aM limit of detection. Applying EPICATCH to quantify MYO1G methylation in clinical plasma cfDNA from colorectal cancer patients yielded a clinical sensitivity of 80.0% and specificity of 84.6%. This dual-zone recognition system provides a robust strategy for precise single-CpG analysis and cancer screening.