TY - JOUR
T1 - An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein
AU - Dewari, Pooran
AU - Southgate, Benjamin
AU - McCarten, Katrina
AU - Monogarov, German
AU - O'Duibhir, Eoghan
AU - Quinn, Niall
AU - Tyrer, Ashley
AU - Leitner, Marie-Christin
AU - Plumb, Colin
AU - Kalantzaki, Maria
AU - Blin, Carla
AU - Finch, Rebecca
AU - Bressan, Raul
AU - Morrison, Gillian
AU - Jacobi, Ashley
AU - Behlke, Mark
AU - von Kriegsheim, Alex
AU - Tomlinson, Simon
AU - Krijgsveld, Jeroen
AU - Pollard, Steven
PY - 2018/4/11
Y1 - 2018/4/11
N2 - 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.
AB - 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.
U2 - 10.7554/eLife.35069
DO - 10.7554/eLife.35069
M3 - Article (Academic Journal)
C2 - 29638216
SN - 2050-084X
VL - 7
JO - eLife
JF - eLife
IS - e35069
ER -