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

Methods : a Companion to Methods in Enzymology
Jan-Hendrik SpilleIbrahim I Cissé

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.

Citations

Oct 17, 2019·Glycobiology·Jonathan SjögrenAndreas Nägeli
Apr 25, 2021·Acta Neuropathologica Communications·M Rebecca GlineburgPeter K Todd
May 1, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Ronja WeissingerOrit Hermesh
Jul 3, 2021·International Journal of Molecular Sciences·Peter HobothPavel Hozák
Feb 18, 2021·Plant & Cell Physiology·Vaishali YadavDurgesh Kumar Tripathi
Jun 16, 2021·Cold Spring Harbor Perspectives in Biology·Jeetayu BiswasRobert A Coleman

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