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

• Published in: Methods (San Diego, Calif.) Calif.), 2019-01, Vol.153, p.35-45
• Main Authors: Spille, Jan-Hendrik, Hecht, Micca, Grube, Valentin, Cho, Won-ki, Lee, Choongman, Cissé, Ibrahim I.
• Format: Article
• Language: English
• Subjects: CRISPR; Live cell imaging; MS2; Quantitative microscopy; Single RNA imaging; Stem cell
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2018.09.004

[Display omitted] •Cloning platform for CRISPR/Cas9-based insertion of MS2 cassette in any coding gene.•Insertion of selectable marker and MS2 cassette in 3′ UTR.•Instructions for verification of correct insertion.•Quantitative fluorescence microscopy of nascent RNA synthesis.•Esrrb down-regulated in binary (all-or-none) manner in mESC differentiation to epiLC. 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.