Programmable RNA Tracking in Live Cells with CRISPR/Cas9

Cell. 2016 Apr 7;165(2):488-96. doi: 10.1016/j.cell.2016.02.054. Epub 2016 Mar 17.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CRISPR-Cas Systems
  • Cytoplasmic Granules / chemistry
  • Endonucleases / metabolism
  • Glyceraldehyde-3-Phosphate Dehydrogenases / genetics
  • Green Fluorescent Proteins / analysis
  • Humans
  • RNA / analysis*
  • RNA, Guide / analysis
  • RNA, Messenger / analysis

Substances

  • RNA, Guide
  • RNA, Messenger
  • Green Fluorescent Proteins
  • RNA
  • Glyceraldehyde-3-Phosphate Dehydrogenases
  • Endonucleases