CbAgo-mediated depletion and Cas9-nickase/sgRNA cleavage triggered exponential amplification reaction for mutant TP53 analysis

Cb Ago selectively depletes wild-type TP53, eliminating background interference for mutant analysis. • Cas9 nickase generates ssDNA initiators to trigger EXPAR under isothermal conditions. • Amplified products disrupt G-quadruplex/ThT fluorescence, enabling a signal-off readout. • The assay achieves 0.14 fM detection limit and single-nucleotide resolution. • Validated in serum and cells; consistent with PCR; discriminates cancer from normal cells. Accurate detection of rare mutant TP53 in the presence of abundant wild-type background remains a major challenge for early diagnosis of colorectal cancer. Here, we report a background-reduced analytical strategy that integrates Cb Ago-mediated wild-type depletion with Cas9 nickase (Cas9n)/sgRNA-triggered exponential amplification reaction (EXPAR) for highly sensitive and specific mutant TP53 analysis. Cb Ago programmed with a guide DNA selectively cleaves wild-type TP53 while preserving the mutant target, thereby eliminating interference from the overwhelming wild-type background. The remaining mutant TP53 is then recognized by two Cas9n/sgRNA complexes, which generate single-stranded initiator fragments through polymerase-assisted strand displacement. These initiators trigger an EXPAR cascade that ultimately disrupts the G-quadruplex structure of a thioflavin T (ThT) fluorescent probe, producing a signal-off readout. The method achieves a detection limit as low as 0.14 fM with excellent single-nucleotide discrimination capability. Its practical utility is validated through recovery experiments, differentiation of cancer cells from normal cells, and high consistency with PCR-based methods in clinical tissue samples. This Cb Ago-Cas9n-EXPAR platform offers a robust and isothermal tool for early diagnosis of TP53-mutated colorectal cancer and can be readily reprogrammed for other clinically relevant single-nucleotide variants. Download: Download high-res image (123KB) Download: Download full-size image