Leading science, pioneering therapies
CRM Publications

An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein.

TitleAn efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein.
Publication TypeJournal Article
Year of Publication2018
AuthorsDewari 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
JournalElife
Volume7
Date Published2018 Apr 11
ISSN2050-084X
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.

DOI10.7554/eLife.35069
Alternate JournalElife
PubMed ID29638216
Grant ListA17368 / / 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 /
Publication institute
CRM