Highly sensitive detection of exosomal miR-122 with CRISPR-Dx for precise HCC diagnosis.

Hepatocellular carcinoma (HCC), one of the most common malignancies worldwide, poses a major threat to the global health. Its prognosis depends heavily on early detection. Currently, alpha-fetoprotein (AFP) is the widely used biomarker for HCC diagnosis. Nevertheless, approximately 40% of the HCC cases do not produce AFP. It has been reported that the downregulation of miR-122 is strongly associated with the development and progression of HCC. Here, employing miR-122 as a biomarker, we developed a cascade amplification-based CRISPR-Dx platform for HCC diagnosis. Owing to the strong trans-cleavage activities of Cas13a and Cas12a, the proposed assay enabled sensitive miR-122 detection down to 1 aM. Clinical sample analysis (n = 48) demonstrated a sensitivity of 91.3%, a specificity of 84.0%, and an overall accuracy of 87.5%, which is significantly better than that of the conventional AFP-based diagnosis (sensitivity, 43.5%; specificity, 80%; accuracy, 62.5%). In addition, because CRISPR-Dx eliminates the RNA extraction and reverse transcription steps that may cause target loss, it also showed better performance than miR-122-targeted qPCR. Given its high diagnostic accuracy and simplified workflow, the proposed miR-122-based CRISPR-Dx assay holds great promise for the clinical diagnosis of HCC.