Cell http://dx.doi.org/10.1016/j.cell.2016.02.054 (2016)

While fluorescent protein fusions are widely used to visualize the cellular localization of proteins, very few methods exist to follow RNA processing and trafficking. Nelles et al. now engineer the CRISPR-Cas9 system, a pervasive tool for targeted genome editing, into a system capable of tracking specific RNAs within living cells. In this RCas9 system, the authors fuse an inactivated Cas9 nuclease (dCas9) to a fluorescent protein, equip it with a single-guide RNA (sgRNA) that targets dCas9 to a specific mRNA sequence, and include a “PAMmer” synthetic oligonucleotide that binds the target mRNA and also prevents DNA targeting. The RCas9 approach was applied to track the movement of several mRNAs, with results that correlated closely with those of RNA fluorescence in situ hybridization (FISH) assays. RCas9–mRNA complexes were sufficiently stable to allow visualization of mRNA transport from the nucleus to the cytoplasm or to follow the trafficking of endogenous mRNAs, such as ACTB, to stress granules after induction of cellular stress. Further, RCas9 expression and RNA binding do not perturb the cellular abundance or function of target mRNAs. Taken together, these results validate RCas9 as a useful tool for RNA visualization that may be readily adapted to probe RNA processing and function within cells.