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Given the fact that the localization of RNAs is closely associated with their functions, techniques developed for tracking the distribution of RNAs in live cells have greatly advanced the study of RNA biology. Recently, innovative application of fluorescent protein-labelled Cas9 and Cas13 into live-cell RNA tracking further enriches the toolbox. However, the Cas9/Cas13 platform, as well as the widely-used MS2-MCP technique, failed to solve the problem of high background noise. It was recently reported that CRISPR/Cas6 would exhibit allosteric alteration after interacting with the Cas6 binding site (CBS) on RNAs. Here, we exploited this feature and designed a Cas6-based switch platform for detecting target RNAs in vivo. Conjugating split-Venus fragments to both ends of the endoribonuclease-mutated Escherichia coli Cas6(dEcCas6) allowed ligand (CBS)-activated split-Venus complementation. We name this platform as Cas6 based Fluorescence Complementation (Cas6FC). In living cells, Cas6FC could detect target RNAs with nearly free background noise. Moreover, as minimal as one copy of CBS (29nt) tagged in an RNA of interest was able to turn on Cas6FC fluorescence, which greatly reduced the odds of potential alteration of conformation and localization of target RNAs. Thus, we developed a new RNA tracking platform inherently with high sensitivity and specificity.

A Cas6-based RNA tracking platform functioning in a fluorescence-activation mode