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

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...

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Published inbioRxiv
Main Authors Pooran Singh Dewari, Southgate, Benjamin, Mccarten, Katrina, Monogarov, German, O'duibhir, Eoghan, Quinn, Niall, Tyrer, Ashley, Plumb, Colin, Blin, Carla, Finch, Rebecca, Raul Bardini Bressan, Morrison, Gillian, Jacobi, Ashley M, Behlke, Mark A, Alex Von Kriegsheim, Tomlinson, Simon, Krijgsveld, Jeroen, Pollard, Steven M
Format Paper
LanguageEnglish
Published Cold Spring Harbor Cold Spring Harbor Laboratory Press 29.01.2018
Subjects
Brain stem
Brain tumors
Chromatin
CRISPR
Epitopes
Localization
Myc protein
Olig2 protein
Pipelines
Proteins
Stem cells
Transcription factors
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Summary: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 sixty 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:10.1101/255737