Abstract
The need to generate modified cell lines that express tagged proteins of interest has become increasingly important. Here, we describe a detailed protocol for facile CRISPR/Cas9-mediated gene tagging and isolation of modified cells. In this protocol, we combine two previously published strategies that promote CRISPR/Cas9-mediated gene tagging: using chemically modified single-stranded oligonucleotides as donor templates and a co-selection strategy targeting the ATP1A1 gene at the same time as the gene of interest. Altogether, the protocol proposed here is both easier and saves time compared to other approaches for generating cells that express tagged proteins of interest, which is crucial to purify native complex from human cells.
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Acknowledgments
This work was supported by the Centre National pour la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Strasbourg (UdS), Association pour la Recherche sur le Cancer (ARC), the Ligue nationale contre le cancer, Institut National du Cancer (INCa; INCA 9378), Agence National pour la Recherche (ANR-12-BSV8-0015-01 and ANR-10-LABX-0030-INRT under the program Investissements d’Avenir ANR-10-IDEX-0002-02), Instruct-ERIC (R&D Project Funding) and by Instruct-ULTRA (Coordination and Support Action Number ID 731005) funded by the EU H2020 framework to further develop the services of Instruct-ERIC.
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Geny, S., Pichard, S., Poterszman, A., Concordet, JP. (2021). Gene Tagging with the CRISPR-Cas9 System to Facilitate Macromolecular Complex Purification. In: Owens, R.J. (eds) Structural Proteomics. Methods in Molecular Biology, vol 2305. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1406-8_8
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DOI: https://doi.org/10.1007/978-1-0716-1406-8_8
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-1406-8
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