Cell
Volume 165, Issue 2, 7 April 2016, Pages 488-496
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Programmable RNA Tracking in Live Cells with CRISPR/Cas9

https://doi.org/10.1016/j.cell.2016.02.054Get rights and content
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Highlights

  • RNA-targeting Cas9 (RCas9) enabled recognition of endogenous, unmodified mRNAs

  • RCas9 did not influence mRNA abundance or amount of translated protein

  • Subcellular distribution of RCas9 was highly correlated with RNA-FISH

  • RCas9 revealed trafficking of mRNAs to stress granules in live cells

Summary

RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA would allow alteration and imaging of endogenous RNA transcripts analogous to CRISPR/Cas-based genomic tools, but most RNA targeting methods rely on incorporation of exogenous tags. Here, we demonstrate that nuclease-inactive S. pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells. We show that nuclear-localized RNA-targeting Cas9 (RCas9) is exported to the cytoplasm only in the presence of sgRNAs targeting mRNA and observe accumulation of ACTB, CCNA2, and TFRC mRNAs in RNA granules that correlate with fluorescence in situ hybridization. We also demonstrate time-resolved measurements of ACTB mRNA trafficking to stress granules. Our results establish RCas9 as a means to track RNA in living cells in a programmable manner without genetically encoded tags.

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