Single-molecule live-cell RNA imaging with CRISPR-Csm

Abstract

AbstractHigh-resolution, real-time imaging of RNA is essential for understanding the diverse, dynamic behaviors of individual RNA molecules in single cells. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved. Here, we present single-molecule live-cell fluorescencein situhybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR-Csm complex with multiplexed guide RNAs for efficient, direct visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we tracked individual endogenousNOTCH2andMAP1BmRNA transcripts in living cells and identified two distinct localization mechanisms: co-translational translocation ofNOTCH2mRNA at the endoplasmic reticulum, and directional transport ofMAP1BmRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.