A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells
Name
Spille_Main_Revision_20180907_FINAL-DSpace.pdf
Description
Accepted version
Size
1.45 MB
Format
Adobe PDF
Checksum (MD5)
b01f4917a94c591f931b282484c9077f
Author(s)
Spille, Jan Hendrik
Hecht, Micca
Grube, Valentin
Cho, Won-ki
Lee, Choongman
Cissé, Ibrahim I.
Date Issued
2019
Journal
Methods
Publisher
Elsevier BV
Citation
Spille, Jan-Hendrik et al. "A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells." Methods 153 (January 2019): 35-45 © 2018 Elsevier Inc
Version
Author's final manuscript
Abstract
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. Keywords: CRISPR; Stem cell; MS2; Single RNA imaging; Quantitative microscopy; Live cell imaging
MIT Department
Massachusetts Institute of Technology. Department of Physics
Massachusetts Institute of Technology. Materials Research Laboratory
Terms of Use
Creative Commons Attribution-NonCommercial-NoDerivs License
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1016/j.ymeth.2018.09.004
