Rapid <i>in situ</i> RNA imaging based on Cas12a thrusting strand displacement reaction
Xiaoxue Cheng, Xiaosong Li, Yuexi Kang, Decai Zhang, Qiubo Yu, Junman Chen, Xinyu Li, Li Du, Tiantian Yang, Yao Gong, Ming Yi, Songzhi Zhang, Shasha Zhu, Shijia Ding, Wei Cheng
Abstract
RNA In situ imaging through DNA self-assembly is advantaged in illustrating its structures and functions with high-resolution, while the limited reaction efficiency and time-consuming operation hinder its clinical application. Here, we first proposed a new strand displacement reaction (SDR) model (Cas12a thrusting SDR, CtSDR), in which Cas12a could overcome the inherent reaction limitation and dramatically enhance efficiency through energy replenishment and by-product consumption. The target-initiated CtSDR amplification was established for RNA analysis, with order of magnitude lower limit of detection (LOD) than the Cas13a system. The CtSDR-based RNA in situ imaging strategy was developed to monitor intra-cellular microRNA expression change and delineate the landscape of oncogenic RNA in 66 clinic tissue samples, possessing a clear advantage over classic in situ hybridization (ISH) in terms of operation time (1 h versus 14 h) while showing comparable sensitivity and specificity. This work presents a promising approach to developing advanced molecular diagnostic tools.