ArticleeLife2018
An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein.
Article in eLife, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 36 papers.
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Who cites it
36 citing papers in PubMed, 66 citations in OpenAlex.
- HIT-MAP: A scalable approach to multimodal mapping of subcellular organization.Cell reports methods · 2026Article
- Brachyury expression levels predict lineage potential and axis-forming ability of in vitro-derived neuromesodermal progenitors.PLoS biology · 2026Article
- CRISPR Gene Tagging for Illuminating Endogenous Protein Dynamics.International journal of molecular sciences · 2026Review
- Review
- Degron models: a toolbox for rapid in vivo depletion of essential proteins regulating mRNA metabolism.Communications biology · 2026Article
- Cell-type-specific functionality encoded within the intrinsically disordered regions of OCT4.Nature communications · 2025Article
- Chromatin-dependent motif syntax defines differentiation trajectories.Molecular cell · 2025Article
- Leveraging CRISPR activation for rapid assessment of gene editing products in human pluripotent stem cells.Stem cell reports · 2025Article
- Intracellular protein editing enables incorporation of noncanonical residues in endogenous proteins.Science (New York, N.Y.) · 2025Article
- Activation-derepression synergy enables a bHLH network to coordinate a signal-specific fate response.Cell reports · 2024Article
- Review
- Modelling quiescence exit of neural stem cells reveals a FOXG1-FOXO6 axis.Disease models & mechanisms · 2024Article
- An Inducible Luminescent System to Explore Parkinson's Disease-Associated Genes.International journal of molecular sciences · 2024Article
- protoSpaceJAM: an open-source, customizable and web-accessible design platform for CRISPR/Cas insertional knock-in.Nucleic acids research · 2024Article
- Article
- Detecting PTP Protein-Protein Interactions by Fluorescent Immunoprecipitation Analysis (FIPA).Methods in molecular biology (Clifton, N.J.) · 2024Article
- MSL2 ensures biallelic gene expression in mammals.Nature · 2023Article
- The Fgf/Erf/NCoR1/2 repressive axis controls trophoblast cell fate.Nature communications · 2023Article
- Article
- Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress.Nature microbiology · 2022Article
Corrections and comments
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Authors and funding
20 authors at 3 institutions in 3 countries.
Funding
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-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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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.