AND logic-gated CRISPR/Cas9 and hybridization chain reaction system for precise ctDNA detection

Circulating tumor DNA (ctDNA) is a critical biomarker for liquid biopsies, enabling the non-invasive acquisition of cancer-related information from blood samples. Precise detection of ctDNA, particularly the identification of single-nucleotide variations (SNVs), is crucial for early cancer diagnosis, therapeutic monitoring, and prognostic evaluation. However, current ctDNA detection methods often encounter challenges such as complex procedures, difficult data analysis, and false-positive signals during pre-amplification. In this study, we introduce a novel detection method based on AND logic-gated integration of interspaced short palindromic repeats and associated proteins (CRISPR/Cas9) system with hybridization chain reaction (HCR) isothermal amplification. This strategy enhances the specific and sensitive detection of ctDNA. The incorporation of the AND logic gate effectively minimizes the off-target effects of Cas9 and enables the differentiation of single-nucleotide mutations, such as KRAS G12D, even in complex serum environments. Our system exhibits high sensitivity and specificity, achieving a limit of detection as low as 1 fM and capable of identifying SNVs mutations with allele fractions as low as 0.1% among wild-type sequences. Furthermore, we validated the specificity of our approach by successfully detecting various mutations, including KRAS G12C, KRAS G12D, EGFR T790M and TP53 R273H, in simulated clinical samples. These findings highlight a reliable method for precise ctDNA detection, offering high specificity, selectivity, and accuracy, thus paving the way for potential cancer diagnostic application.