CRISPR Cas12a-mediated amplification-free digital DNA assay improves the diagnosis and surveillance of Nasopharyngeal carcinoma

Sensitive and accurate cell-free plasma Epstein-Barr virus (EBV) DNA measurement is essential in the routine diagnosis, monitoring and treatment of Nasopharyngeal Carcinoma (NPC). This measurement in commercial and in-house assay are commonly based on real-time quantitative PCR (qPCR) method, which requires reference materials for standardization and lack quantitative precision due to amplification bias or cross-contamination. To address these issues, we developed a CRISPR/Cas12a-mediated amplification-free digital DNA assay, which targets the repetitive sequences of EBV DNA and utilizes the cis-cleavage activity of CRISPR-Cas12a prior to droplet generation. By this mean, more activated Cas12a-crRNA duplexes could be produced for subsequent target detection and counting, thus improving the performance in detecting low EBV DNA load. We demonstrated that it was more robust than conventional qPCR for detecting plasma EBV DNA in a case-control study of 208 participants, especially when the target concentrations were around the diagnostic cut-off value for NPC. More importantly, this assay allowed a more accurate diagnosis of early-stage NPC, with an area under the curve (AUC) of 0.9883 (versus 0.7682 for qPCR). Furthermore, its absolute quantification capability enabled dynamic monitoring of EBV load in NPC patients during initial diagnosis, treatment, and recurrence, thereby potentially improving disease management and prognosis. Taken together, our results demonstrate that this amplificationfree digital assay has the potential to be a robust tool to improve the diagnosis and surveillance of NPC.