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.
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
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