Signal logic gate mediated by a controllable CRISPR/Cas12a system for simultaneous detection of DNA mutation and methylation.

Early detection of both genetic mutations and epigenetic modifications is critical for cancer diagnosis, but current methods often require separate assays or suffer from bisulfite-induced DNA damage. Here, we present a controllable CRISPR/Cas12a-based signal logic gate that enables simultaneous detection of KRAS G12C mutation and Septin9 promoter methylation in a single reaction. The strategy converts target information into two distinct ssDNA activators (T1 for methylation and T2 for mutation) via orthogonal enzymatic cascades (GlaI- and FEN1-mediated cleavage followed by strand displacement amplification). By limiting crRNA concentrations, the CRISPR/Cas12a trans-cleavage activity produces three well-resolved fluorescence kinetic states: low (mutation only), medium (methylation only), and high (both targets), respectively, which could be distinguished using three predefined threshold values. Validation with clinical samples from colorectal cancer patients and healthy controls showed complete concordance with Sanger sequencing and qPCR. And the proposed method successfully detected Septin9 methylation in peripheral blood. This isothermal, single-tube, bisulfite-free strategy offers a simple and reliable platform for simultaneous genetic and epigenetic analysis in point-of-care cancer screening.