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

Spille, Jan-Hendrik and Hecht, Micca and Grube, Valentin and Cho, Won-ki and Lee, Choongman and Cissé, Ibrahim I. (2019) A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells. Methods, 153 . pp. 35-45. ISSN 1046-2023. https://resolver.caltech.edu/CaltechAUTHORS:20201112-143949737

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.ymeth.2018.09.004DOIArticle
ORCID:
AuthorORCID
Spille, Jan-Hendrik0000-0001-8493-4721
Hecht, Micca0000-0002-0057-1554
Cissé, Ibrahim I.0000-0002-8764-1809
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.
Funders:
Funding AgencyGrant Number
NIHDP2CA195769
Deutsche Forschungsgemeinschaft (DFG)SP1680/1-1
Subject Keywords:CRISPR; Stem cell; MS2; Single RNA imaging; Quantitative microscopy; Live cell imaging
Record Number:CaltechAUTHORS:20201112-143949737
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20201112-143949737
Official Citation:Jan-Hendrik Spille, Micca Hecht, Valentin Grube, Won-ki Cho, Choongman Lee, Ibrahim I. Cissé, A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells, Methods, Volume 153, 2019, Pages 35-45, ISSN 1046-2023, https://doi.org/10.1016/j.ymeth.2018.09.004.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:106661
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:16 Nov 2020 16:00
Last Modified:16 Nov 2020 16:00

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