Thermosensitive hydrogel-enhanced RPA-CRISPR/Cas12a biosensor for ultrasensitive detection of methylated loci in breast cancer ctDNA.

[BACKGROUND] Methylation differences exist between breast cancer tissues and normal tissues. The release of methylated circulating tumor DNA (ctDNA) by tumor cells provides a foundation for breast cancer liquid biopsy using methylated ctDNA. However, detection of low-abundance methylated loci in ctDNA remains a significant challenge to date. Existing one-tube detection systems cannot avoid target depletion caused by CRISPR/Cas12a cleavage, leading to reduced sensitivity.

[RESULTS] This study developed a thermosensitive hydrogel-based one-tube RPA-CRISPR/Cas12a detection system, combined with methylation-sensitive restriction endonucleases (MSRE), for the detection of specific methylated loci in breast cancer ctDNA. This method achieves spatial separation while maintaining connectivity of reaction phases in a single tube for the first time, and the thermosensitive hydrogel does not exert inhibitory effects on either system. The system can specifically recognize methylated target molecules with a limit of detection (LOD) as low as 1 × 10 ng/μL (≈70 copies/μL), outperforming the current glycerol-enhanced one-tube reaction system. It is capable of distinguishing methylated fractions as low as 0.05 %, with a sensitivity twice that of the gold standard methylation-specific quantitative PCR (Methylight). Detection of genomic DNA (gDNA) from tumor tissues and paired plasma ctDNA of 15 clinical patients using this method showed both sensitivity and specificity reaching 100 %.

[SIGNIFICANCE] This novel, highly sensitive, efficient, and portable detection method innovatively resolves the target depletion issue caused by CRISPR/Cas12a cleavage in traditional RPA-CRISPR/Cas12a systems via thermosensitive hydrogel-mediated single-tube phase separation technology. It provides a new technical pathway for the accurate detection of low-abundance methylated circulating tumor DNA (ctDNA), will significantly enhance the level of breast cancer liquid biopsy, and offer strong support for the diagnosis and differential diagnosis of breast cancer.