Title | An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein. |
Publication Type | Journal Article |
Year of Publication | 2018 |
Authors | Dewari PSingh, Southgate B, Mccarten K, Monogarov G, O'Duibhir E, Quinn N, Tyrer A, Leitner M-C, Plumb C, Kalantzaki M, Blin C, Finch R, Bressan RBardini, Morrison G, Jacobi AM, Behlke MA, von Kriegsheim A, Tomlinson SR, Krijgsveld J, Pollard SM |
Journal | Elife |
Volume | 7 |
Date Published | 2018 Apr 11 |
ISSN | 2050-084X |
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. |
DOI | 10.7554/eLife.35069 |
Alternate Journal | Elife |
PubMed ID | 29638216 |
Grant List | A17368 / / Cancer Research UK / United Kingdom BB/M018040/1 / / Medical Research Council / United Kingdom BB/M018040/1 / / Biotechnology and Biological Sciences Research Council / United Kingdom BB/M018040/1 / / Engineering and Physical Sciences Research Council / GN-000358 / / Brain Tumour Charity / |