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. doi:10.1016/j.ymeth.2018.09.004. https://resolver.caltech.edu/CaltechAUTHORS:20201112-143949737
![[img]](https://authors.library.caltech.edu/style/images/fileicons/text_xml.png) |
XML (Supplementary data 1)
- Supplemental Material
See Usage Policy. 244B |
![[img]](https://authors.library.caltech.edu/style/images/fileicons/video.png) |
Video (MPEG) (Supplementary video 2)
- Supplemental Material
See Usage Policy. 1MB |
Use this Persistent URL to link to this item: https://resolver.caltech.edu/CaltechAUTHORS:20201112-143949737
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: |
| ||||||||
| ORCID: |
| ||||||||
| 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: |
| ||||||||
| Subject Keywords: | CRISPR; Stem cell; MS2; Single RNA imaging; Quantitative microscopy; Live cell imaging | ||||||||
| DOI: | 10.1016/j.ymeth.2018.09.004 | ||||||||
| 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 2021 18:55 |
Repository Staff Only: item control page
