RESEARCH HIGHLIGHTS

SENSORS AND PROBES Hijacking Cas9 for live-cell RNA imaging

Researchers target fluorescent protein– motif (PAM) that resides next to the target Cas9 fusions to specific mRNAs for track- site, but on the opposite strand, for bind- ing in live cells. ing. Instead, Yeo’s team introduced the RNAs are a vitally important class of bio- PAM in trans to the target single-stranded molecules that function at every level of RNA using a DNA oligonucleotide (known gene expression. However, tools for imag- as a PAMmer). The team also had to use a ing RNA in living cells lag behind analogous mutant of Cas9 that is enzymatically inac- methods for imaging protein abundance, tive and a chemically modified PAMmer to localization and dynamics. The classic avoid RNA cleavage. Finally, they tagged approach for imaging RNA abundance and Cas9 with a fluorescent protein to light up localization in cells is fluorescence in situ the RNAs of interest for imaging. hybridization (FISH). Although powerful, In their first set of experiments, the Cellular RNA imaged with FISH (red) and RCas9– FISH requires cell fixation, which is not researchers demonstrated that Cas9– GFP (green). Blue signal represents nuclear DNA. Adapted from Nelles et al. (2016) with permission compatible with live-cell imaging. However, mCherry could specifically recognize an from Elsevier. most tools for live-cell RNA imaging involve mRNA in the cell’s nucleus and then be genetically tagging the RNA of interest. properly exported into the cytoplasm along The team also tested whether imaging Gene Yeo and his graduate student David with the RNA. These experiments also with RNA-targeting Cas9 (RCas9) affects Nelles at the University of California, San revealed that the sgRNA was the key player the stability of the targeted mRNA or the Diego, sought to overcome these limitations in RNA targeting, but that the PAMmer also expression of the encoded protein. In inves- using CRISPR/Cas9 technology, which has contributed to binding efficiency. The team tigations of both questions, they saw no become wildly popular for its use in targeted then demonstrated that targeting is spe- change in mRNA or protein levels upon genome editing. Commonly used CRISPR/ cific by showing colocalization with FISH targeting, indicating that these factors are

Nature America, Inc. All rights reserved. America, Inc. © 201 6 Nature Cas9 systems are composed of two key ele- against the same mRNAs in cells. They note not perturbed by this labeling strategy. ments: the Cas9 protein and a single guide that these experiments in particular were The potential applications of RCas9 are RNA (sgRNA). Cas9 binds to the sgRNA, technically challenging because they used seemingly boundless. Yeo says that his and the complex then targets a site of inter- different modalities of RNA recognition team is working hard to improve the sensi- npg est in the genome determined by comple- and signal amplification. They also found tivity and resolution of the technology for mentarity to that site in the sgRNA. the results to be better than expected. “Dave RNA localization, and they are also inter- In most circumstances, the CRISPR/Cas9 and I were surprised that a single dCas9– ested in identifying other Cas proteins that system is used to target DNA. However, Yeo GFP was able to correlate reasonably with can be used for RNA recognition. Such recalls, “my lab came to the quick realiza- RNA FISH measurements which typically proteins could be useful tools for multi- tion that the Cas9–sgRNA complex may involve tens to dozens (sometimes up to 50) plexed live-cell RNA imaging. In addition, be hijacked for RNA recognition.” He also of probes,” explains Yeo. outside of imaging, RCas9 could be used to notes that by the time they had begun their The researchers also demonstrated the site-specifically target proteins of interest work, “our friend and colleague Jennifer power of this method for live-cell imag- to RNAs to allow scientists to study their Doudna showed that the complex could ing by monitoring mRNA trafficking into functions. This work should also move Yeo recognize RNA in vitro.” So they began stress granules in cells undergoing oxida- and his team closer to other research goals, collaborating with Doudna and Mitchell tive stress. In those experiments they indi- such as understanding how RNA localiza- O’Connell, a postdoc in her laboratory, to vidually monitored three different mRNA tion and transport are involved in proper develop the system further for recognition molecules during stress using Cas9–GFP in health and in neurological diseases such as of RNA in live cells. cells that also expressed a marker for stress amyotrophic lateral sclerosis. Their method involves targeting Cas9 granules. In all cases, the mRNAs, which Rita Strack to RNA rather than DNA. Under normal were initially distributed in the cytoplasm, RESEARCH PAPERS circumstances, Cas9 is specific for DNA became localized over time in the stress Nelles, D.A. et al. Programmable RNA tracking in live because it requires a protospacer-adjacent granules. cells with CRISPR/Cas9. Cell 165, 1–9 (2016).

NATURE METHODS | VOL.13 NO.5 | MAY 2016 | 391