The development of precise RNA editing tools is essential for the advancement of RNA therapeutics. The discovery of RNA-guided RNA-targeting Cas effectors called CRISPR-Cas13 (Cas13) has revolutionised the field of RNA editing. These enzymes are readily programmable for the recognition and silencing of any transcripts of interest in a sequence-specific manner. The design flexibility of Cas13 platforms offers a great opportunity to target “undruggable” genes that drive various diseases including cancer. However, the mechanisms that govern Cas13 target search in the crowded and highly compartmentalised cellular environment of human cells remain elusive, limiting the use of these programmable platforms in precise transcriptome editing.
In this study, we employed a combination of in vitro and in vivo biochemical assays and live-cell imaging to elucidate the mechanisms that govern Cas13 target search in live cells. Our findings suggest that the spatial colocalization of Cas13 and the target RNA in specific subcellular compartments can define Cas13 targeting efficiency. We have further established a high-resolution map of RNA molecules that are either prone or resilient to Cas13 silencing. These findings have important implications for the engineering of Cas13 for the silencing of previously untargetable transcripts. Overall, this study provides a foundation for the development of more effective Cas13-based RNA editing tools for precise and potent silencing of various pathogenic transcripts including oncogenic RNA.