DNA mutation and methylation detected at the same time using a controllable CRISPR-based signal system

A controllable CRISPR/Cas12a-based signal logic gate enables simultaneous detection of KRAS G12C mutation and Septin9 promoter methylation in a single reaction.

• The method converts target information into two distinct single-stranded DNA activators for methylation and mutation.
• Three fluorescence kinetic states are produced: low (mutation only), medium (methylation only), and high (both targets).
• Validation with clinical samples from colorectal cancer patients and healthy controls showed complete agreement with Sanger sequencing and qPCR.
• The method successfully detected Septin9 methylation in peripheral blood.
• This approach is isothermal, single-tube, and does not require bisulfite treatment.

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