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

Pooran Dewari, Benjamin Southgate, Katrina McCarten, German Monogarov, Eoghan O'Duibhir, Niall Quinn, Ashley Tyrer, Marie-Christin Leitner, Colin Plumb, Maria Kalantzaki, Carla Blin, Rebecca Finch, Raul Bressan, Gillian Morrison, Ashley Jacobi, Mark Behlke, Alex von Kriegsheim, Simon Tomlinson, Jeroen Krijgsveld, Steven Pollard

Research output: Contribution to journalArticle (Academic Journal)peer-review

28 Citations (Scopus)
307 Downloads (Pure)


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-tumor-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.
Original languageEnglish
Issue numbere35069
Publication statusPublished - 11 Apr 2018


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