An Exosome RNA In Situ Detection Platform Based on a Regulated CRISPR/Cas12a Activity System and Its Application in Tumor Progression Monitoring and Therapeutic Efficacy Evaluation

Abstract

Tumor-derived exosomes, serving as promising biomarkers, hold substantial potential for reflecting the progression of diseases and assessing the efficacy of antitumor therapies. In this study, we developed a liposome-based platform capable of both precise recognition of tumor-derived exosomes and highly sensitive in situ analysis of the internal RNA. Leveraging surface-anchored DNA tags and their hybridization with two allosteric aptamers targeting exosomal marker CD63 and tumor marker PD-L1, the platform can specifically capture tumor-derived exosomes and facilitate the membrane fusion. Upon mixing of the vesicular contents, an internal functional duplex converter undergoes strand displacement with the target mRNA, releasing an uncaged strand. This strand eliminates the inhibition of the Cas12a/crRNA assembly imposed by an elongation-caged activator, thereby restoring the transcleavage activity of Cas12a. The activated Cas12a cleaves reporter probes to generate a fluorescent signal, enabling highly sensitive in situ detection of tumor-derived exosomal RNA. The constructed platform enables dynamic monitoring of disease progression in tumor-bearing mice by quantifying the relative levels of tumor-derived exosomes in serum and further distinguishes therapeutic outcomes among different drug treatments. This study highlights the significant potential of the proposed platform in tumor diagnosis and the evaluation of antitumor therapeutic efficacy.