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.
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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
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High-throughput vertebrate animal model systems for the study of patient-specific biology and new therapeutic approaches for aggressive brain tumors are currently lacking, and new approaches are urgently needed. Therefore, to build a patient-relevant in vivo model of human glioblastoma, we expressed common oncogenic variants including activated human EGFRvIII and PI3KCAH1047R under the control of the radial glial-specific promoter her4.1 in syngeneic tp53 loss-of-function mutant zebrafish. Robust tumor formation was observed prior to 45 days of life, and tumors had a gene expression signature similar to human glioblastoma of the mesenchymal subtype, with a strong inflammatory component. Within early stage tumor lesions, and in an in vivo and endogenous tumor microenvironment, we visualized infiltration of phagocytic cells, as well as internalization of tumor cells by mpeg1.1:EGFP+ microglia/macrophages, suggesting negative regulatory pressure by pro-inflammatory cell types on tumor growth at early stages of glioblastoma initiation. Furthermore, CRISPR/Cas9-mediated gene targeting of master inflammatory transcription factors irf7 or irf8 led to increased tumor formation in the primary context, while suppression of phagocyte activity led to enhanced tumor cell engraftment following transplantation into otherwise immune-competent zebrafish hosts. Altogether, we developed a genetically relevant model of aggressive human glioblastoma and harnessed the unique advantages of zebrafish including live imaging, high-throughput genetic and chemical manipulations to highlight important tumor-suppressive roles for the innate immune system on glioblastoma initiation, with important future opportunities for therapeutic discovery and optimizations.
