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

• Main Authors: Spille, Jan Hendrik (Author), Hecht, Micca (Author), Grube, Valentin (Author), Cho, Won-ki (Author), Lee, Choongman (Author), Cissé, Ibrahim I. (Author)
• Other Authors: Massachusetts Institute of Technology. Department of Physics (Contributor), Massachusetts Institute of Technology. Materials Research Laboratory (Contributor)
• Format: Article
• Language: English
• Published: Elsevier BV, 2020-02-05T20:07:37Z.
• Subjects: Article
• Online Access: Get fulltext

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