Multiple RNA Rapid In Situ Imaging Based on Cas9 Code Key System

Abstract Existing RNA in situ imaging strategies mostly utilize parallel repetitive nucleic acid self‐assembly to achieve multiple analysis, with limitations of complicated systems and cumbersome steps. Here, a Cas9 code key system with key probe (KP) encoder and CRISPR/Cas9 signal exporter is developed. This system triggers T‐protospacer adjacent motif (T‐PAM structural transitions of multiple KP encoders to form coding products with uniform single‐guide RNA (sgRNA) target sequences as tandem nodes. Only single sgRNA/Cas9 complex is required to cleave multiple coding products, enabling efficient “many‐to‐one” tandem signaling, and non‐collateral cleavage activity‐dependent automatic signaling output through active introduction of mismatched bases. Compared with conventional parallel multiple signaling analysis model, the proposed system greatly simplifies reaction process and enhances detection efficiency. Further, a rapid multiple RNA in situ imaging system is developed by combining the Cas9 code key system with a T‐strand displacement amplification (T‐SDA) signal amplifier. The constructed system is applied to tumor cells and clinicopathology slices, generating clear multi‐mRNA imaging profiles in less than an hour with just one step. Therefore, this work provides reliable technical support for clinical tumor typing and molecular mechanism investigation.