A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cell

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The MS2 system is a powerful tool for investigating transcription dynamics at the single molecule directly in live cells. In the past, insertion of the RNA-labelling cassette at specific gene loci has been a major hurdle. Here, we present a CRISPR/Cas9-based approach to insert an MS2 cassette with selectable marker at the start of the 3' untranslated region of any coding gene. We demonstrate applicability of our approach by tagging RNA of the stem cell transcription factor Esrrb in mouse embryonic stem cells. Using quantitative fluorescence microscopy we determine the number of nascent transcripts at the Esrrb locus and the fraction of cells expressing the gene. We find that upon differentiation towards epiblast-like cells, expression of Esrrb is down-regulated in an increasing fraction of cells in a binary manner.
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Issue Date
2019-01
Language
English
Article Type
Article
Citation

METHODS, v.153, pp.35 - 45

ISSN
1046-2023
DOI
10.1016/j.ymeth.2018.09.004
URI
http://hdl.handle.net/10203/268726
Appears in Collection
BS-Journal Papers(저널논문)
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