Journal
ELIFE
Volume 7, Issue -, Pages -Publisher
ELIFE SCIENCES PUBLICATIONS LTD
DOI: 10.7554/eLife.35069
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Funding
- Cancer Research UK [A17368]
- Medical Research Council [BB/M018040/1]
- Biotechnology and Biological Sciences Research Council [BB/M018040/1]
- Engineering and Physical Sciences Research Council [BB/M018040/1]
- Brain Tumour Charity [GN-000358]
- BBSRC [BB/M018040/1] Funding Source: UKRI
- MRC [MR/K017047/1] Funding Source: UKRI
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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.
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