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Programmable RNA Tracking in Live Cells with CRISPR/Cas9
#MMPMID26997482
Nelles DA
; Fang MY
; O'Connell MR
; Xu JL
; Markmiller SJ
; Doudna JA
; Yeo GW
Cell
2016[Apr]; 165
(2
): 488-96
PMID26997482
show ga
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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