Programmable RNA Tracking in Live Cells with CRISPR/Cas9

Abstract

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.

RNA Localization in Bacteria

“…Recently, CRISPR-Cas systems have been engineered to label both DNA (128–130) and RNA (131, 132) in live cells. An early example of RNA labeling with CRISPR-Cas systems originated from the finding that Cas9, an RNA-guided DNase, can also target single-stranded RNA by providing the PAM (protospacer adjacent motif) as part of an oligonucleotide (PAMmer) (133).…”

“…An early example of RNA labeling with CRISPR-Cas systems originated from the finding that Cas9, an RNA-guided DNase, can also target single-stranded RNA by providing the PAM (protospacer adjacent motif) as part of an oligonucleotide (PAMmer) (133). Thereby, a catalytically inactive Cas9 (dCas9), fused to a fluorescent protein and in complex with the PAMmer and single guide RNA (sgRNA), can target and label the RNA of interest in a programmable fashion (131). Recently, Cas13a, an RNA-guided RNA-targeting CRISPR–Cas effector, has been applied for RNA tracking in mammalian cells (132).…”

State of the art technologies to explore long non‐coding RNAs in cancer

“…A nuclease null Cas9 protein fused to EGFP (dCas9‐EGFP) was shown to successfully recognize ACTB, CCNA2 and TFRC mRNAs only in the presence of gRNA 53. However, dCas9 binding affinity to RNA was shown to be augmented in the presence of the PAMmer sequence 52, 53.…”

Induced Pluripotent Stem Cells Meet Genome Editing

“…Moreover, dCas9 fused to fluorescent reporters have been developed to indicate nuclear organization by visualizing individual genomic loci (Chen et al, 2013; Gilbert et al, 2014; Tanenbaum et al, 2014). Excitingly more applications are being developed; recently Cas9 has now also been programmed to target RNA in vitro and in vivo (O’Connell et al, 2014) (Nelles et al, 2016), raising the possibly that it could be used to better understand the transcriptome in addition to the genome.…”