Diagnóstico de cáncer sin amplificación basado en la inhibición de la actividad Cas12a por cfDNA modificado con 5mC...

DNA methylation detection holds significant value for cancer diagnosis and recurrence monitoring. However, current methods are often time-consuming, costly, and necessitate specialized techniques. The CRISPR-Cas system, particularly Cas12a, presents a precise and user-friendly platform for disease diagnosis. We developed the CRISPR-Methylated DNA Detection Test (CRISPR-MeDNA Test), a Cas12a-based method for detecting methylation in plasma cell-free DNA (cfDNA). The results reveal that 5mC-modified DNA significantly suppresses the trans-cleavage activity of Cas12a, depending on the methylation site, number, and interval spacing. Simultaneously, methylation of the non-target strand (NTS) suppresses Cas12a activity more strongly than methylation of the target strand (TS), as the NTS plays a critical role in R-loop formation, which is essential for Cas12a cleavage target DNA. Mechanistically, 5mC-modified DNA was found to trigger conformational rearrangements in the Cas12a complex, as predicted by AlphaFold3 modeling and corroborated by FRET assays. Notably, the combination of Cas12a with multiplexed guide RNAs enables effective discrimination between cfDNA from healthy donors and cancer patients without the need for pre-amplification, based on the inhibitory effects of methylated DNA on the Cas12a trans-cleavage activity. This work provides a Cas12a-based detection for a rapid, cost-effective, low-complexity method for 5mC-modified cfDNA in liquid biopsies.