A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells
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.
Additional Information
© 2018 Elsevier. Received 4 July 2018, Revised 7 September 2018, Accepted 9 September 2018, Available online 12 September 2018. We thank E. Calo (MIT) for the wild-type R1 cells, and differentiation protocol and L. D. Lavis (HHMI/Janelia) and J. Grimm (HHMI/Janelia) for gift of the JF646-SNAP ligand. pCRISPaint-HaloTag-PuroR was a gift from Veit Hornung (Addgene plasmid # 80960). pDZ415 (24MS2SL loxP-Kan-loxP) was a gift from Robert Singer & Daniel Zenklusen (Addgene plasmid # 45162). pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988). This work was supported by the NIH Director's New Innovator award #DP2CA195769 (to I.I.C). J.-H.S. is supported by a postdoctoral fellowship from the German Research Foundation (DFG, SP1680/1-1). Declarations of interest: None.Attached Files
Supplemental Material - 1-s2.0-S104620231830094X-mmc1.xml
Supplemental Material - 1-s2.0-S104620231830094X-mmc2.mp4
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Additional details
- Eprint ID
- 106661
- Resolver ID
- CaltechAUTHORS:20201112-143949737
- NIH
- DP2CA195769
- Deutsche Forschungsgemeinschaft (DFG)
- SP1680/1-1
- Created
-
2020-11-16Created from EPrint's datestamp field
- Updated
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2021-11-16Created from EPrint's last_modified field