An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein
- University of Edinburgh, United Kingdom
- German Cancer Research Center (DKFZ), Germany
- Integrated DNA Technologies, Inc, United States
Abstract
CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5-30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain tumour-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Newly generated cell lines will be made available on request.
Article and author information
Author details
Raul Bardini Bressan
Funding
Cancer Research UK (A17368)
- Pooran Singh Dewari
- Benjamin Southgate
- Eoghan O'Duibhir
- Steven M Pollard
Medical Research Council (BB/M018040/1)
- Pooran Singh Dewari
- Steven M Pollard
Biotechnology and Biological Sciences Research Council (BB/M018040/1)
- Pooran Singh Dewari
- Steven M Pollard
Engineering and Physical Sciences Research Council (BB/M018040/1)
- Pooran Singh Dewari
- Steven M Pollard
Brain Tumour Charity (GN-000358)
- Pooran Singh Dewari
- Steven M Pollard
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Maarten van Lohuizen, The Netherlands Cancer Institute, Netherlands
Version history
- Received: January 12, 2018
- Accepted: April 10, 2018
- Accepted Manuscript published: April 11, 2018 (version 1)
- Version of Record published: May 11, 2018 (version 2)
Copyright
© 2018, Dewari et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 10,963
- views
-
- 1,893
- downloads
-
- 43
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein
eLife 7:e35069.
https://doi.org/10.7554/eLife.35069
Further reading
-
- Cell Biology
- Neuroscience
Mutations in Drosophila Swiss Cheese (SWS) gene or its vertebrate orthologue Neuropathy Target Esterase (NTE) lead to progressive neuronal degeneration in flies and humans. Despite its enzymatic function as a phospholipase is well-established, the molecular mechanism responsible for maintaining nervous system integrity remains unclear. In this study, we found that NTE/SWS is present in surface glia that forms the blood-brain-barrier (BBB) and that NTE/SWS is important to maintain its structure and permeability. Importantly, BBB glia-specific expression of Drosophila NTE/SWS or human NTE in the sws mutant background fully rescues surface glial organization and partially restores BBB integrity, suggesting a conserved function of NTE/SWS. Interestingly, sws mutant glia showed abnormal organization of plasma membrane domains and tight junction rafts accompanied by the accumulation of lipid droplets, lysosomes, and multilamellar bodies. Since the observed cellular phenotypes closely resemble the characteristics described in a group of metabolic disorders known as lysosomal storage diseases (LSDs), our data established a novel connection between NTE/SWS and these conditions. We found that mutants with defective BBB exhibit elevated levels of fatty acids, which are precursors of eicosanoids and are involved in the inflammatory response. Also, as a consequence of a permeable BBB, several innate immunity factors are upregulated in an age-dependent manner, while BBB glia-specific expression of NTE/SWS normalizes inflammatory response. Treatment with anti-inflammatory agents prevents the abnormal architecture of the BBB, suggesting that inflammation contributes to the maintenance of a healthy brain barrier. Considering the link between a malfunctioning BBB and various neurodegenerative diseases, gaining a deeper understanding of the molecular mechanisms causing inflammation due to a defective BBB could help to promote the use of anti-inflammatory therapies for age-related neurodegeneration.
-
- Cell Biology
- Structural Biology and Molecular Biophysics
Mutations in the human PURA gene cause the neurodevelopmental PURA syndrome. In contrast to several other monogenetic disorders, almost all reported mutations in this nucleic acid-binding protein result in the full disease penetrance. In this study, we observed that patient mutations across PURA impair its previously reported co-localization with processing bodies. These mutations either destroyed the folding integrity, RNA binding, or dimerization of PURA. We also solved the crystal structures of the N- and C-terminal PUR domains of human PURA and combined them with molecular dynamics simulations and nuclear magnetic resonance measurements. The observed unusually high dynamics and structural promiscuity of PURA indicated that this protein is particularly susceptible to mutations impairing its structural integrity. It offers an explanation why even conservative mutations across PURA result in the full penetrance of symptoms in patients with PURA syndrome.
