Cancer-Associated Nucleic Acid Detection via One-pot CRISPR-Based Diagnostics

Abstract

Early cancer detection and personalized monitoring require accessible molecular diagnostics that can be performed repeatedly across multiple clinical time points. However, conventional cancer-associated nucleic acid detection methods often depend on centralized laboratory infrastructure, limiting their accessibility for repeated testing. In this study, we evaluated CRISPR-based isothermal diagnostic strategies for detecting two cancer-associated nucleic acid targets with distinct molecular characteristics: miRNA as a candidate target for early cancer detection and ecDNA breakpoint sequences as candidate tumor-informed targets for personalized monitoring. For miRNA detection, a poly（A）-assisted SHARP–CRISPR strategy was designed to convert short miRNA targets into amplifiable cDNA templates, and the sequential operation of each reaction step supported the feasibility of further development toward one-pot implementation. For ecDNA detection, breakpoint sequences distinguishable from chromosomal DNA were selected as ecDNA-specific targets, and one-pot RPA–CRISPR detected the ecDNA breakpoint target down to 0.1 pg/μL total DNA, showing a minimum detectable total DNA concentration comparable to that of qPCR. Taken together, this study supports the feasibility of adapting CRISPR-based isothermal detection strategies to cancer-associated nucleic acid targets with different molecular properties. These findings suggest that CRISPR-based isothermal diagnostics may provide an accessible approach for repeated molecular assessment in early cancer detection and personalized monitoring.