Abstract
Macromolecular complexes govern the majority of biological processes and are of great biomedical relevance as factors that perturb interaction networks underlie a number of diseases, and inhibition of protein–protein interactions is a common strategy in drug discovery. Genome editing technologies enable precise modifications in protein coding genes in mammalian cells, offering the possibility to introduce affinity tags or fluorescent reporters for proteomic or imaging applications in the bona fide cellular context. Here we describe a streamlined procedure which uses the CRISPR/Cas9 system and a double-stranded donor plasmid for efficient generation of homozygous endogenously GFP-tagged human cell lines. Establishing cellular models that preserve native genomic regulation of the target protein is instrumental to investigate protein localization and dynamics using fluorescence imaging but also to affinity purify associated protein complexes using anti-GFP antibodies or nanobodies.
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References
Villicana C, Cruz G, Zurita M (2014) The basal transcription machinery as a target for cancer therapy. Cancer Cell Int 14(1):18
Scott DE, Bayly AR, Abell C, Skidmore J (2016) Small molecules, big targets: drug discovery faces the protein-protein interaction challenge. Nat Rev Drug Discov 15(8):533–550
Bushweller JH (2019) Targeting transcription factors in cancer—from undruggable to reality. Nat Rev Cancer 19(11):611–624
Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1258096
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821
Dalvai M, Loehr J, Jacquet K, Huard CC, Roques C, Herst P, Cote J, Doyon Y (2015) A scalable genome-editing-based approach for mapping multiprotein complexes in human cells. Cell Rep 13(3):621–633
Dewari PS, Southgate B, McCarten K, Monogarov G, O’Duibhir E, Quinn N, Tyrer A, Leitner MC, Plumb C, Kalantzaki M, Blin C, Finch R, Bressan RB, Morrison G, Jacobi AM, Behlke MA, von Kriegsheim A, Tomlinson S, Krijgsveld J, Pollard SM (2018) An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein. elife 7
Geny S, Pichard S, Poterszman A, Concordet J (in press) Gene tagging with the CRISPR-Cas9 system to facilitate macromolecular complex purification. Methods Mol Biol
Compe E, Egly JM (2016) Nucleotide excision repair and transcriptional regulation: TFIIH and beyond. Annu Rev Biochem 85:265–290
Kolesnikova O, Radu L, Poterszman A (2019) TFIIH: a multi-subunit complex at the cross-roads of transcription and DNA repair. Adv Protein Chem Struct Biol 115:21–67
Sandoz J, Nagy Z, Catez P, Caliskan G, Geny S, Renaud JB, Concordet JP, Poterszman A, Tora L, Egly JM, Le May N, Coin F (2019) Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression. Nat Commun 10(1):1288
Yeh CD, Richardson CD, Corn JE (2019) Advances in genome editing through control of DNA repair pathways. Nat Cell Biol 21(12):1468–1478
Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kuhn R (2015) Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol 33(5):543–548
Sluch VM, Chamling X, Wenger C, Duan Y, Rice DS, Zack DJ (2018) Highly efficient scarless knock-in of reporter genes into human and mouse pluripotent stem cells via transient antibiotic selection. PLoS One 13(11):e0201683
Sentmanat MF, Peters ST, Florian CP, Connelly JP, Pruett-Miller SM (2018) A survey of validation strategies for CRISPR-Cas9 editing. Sci Rep 8(1):888
Vouillot L, Thelie A, Pollet N (2015) Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 (Bethesda) 5(3):407–415
Renaud JB, Boix C, Charpentier M, De Cian A, Cochennec J, Duvernois-Berthet E, Perrouault L, Tesson L, Edouard J, Thinard R, Cherifi Y, Menoret S, Fontaniere S, de Croze N, Fraichard A, Sohm F, Anegon I, Concordet JP, Giovannangeli C (2016) Improved genome editing efficiency and flexibility using modified oligonucleotides with TALEN and CRISPR-Cas9 nucleases. Cell Rep 14(9):2263–2272
Shan Q, Wang Y, Li J, Gao C (2014) Genome editing in rice and wheat using the CRISPR/Cas system. Nat Protoc 9(10):2395–2410
Kim HJ, Lee HJ, Kim H, Cho SW, Kim JS (2009) Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome Res 19(7):1279–1288
Guschin DY, Waite AJ, Katibah GE, Miller JC, Holmes MC, Rebar EJ (2010) A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol 649:247–256
Brinkman EK, Chen T, Amendola M, van Steensel B (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42(22):e168
Paix A, Rasoloson D, Folkmann A, Seydoux G (2019) Rapid tagging of human proteins with fluorescent reporters by genome engineering using double-stranded DNA donors. Curr Protoc Mol Biol 129(1):e102
Paix A, Schmidt H, Seydoux G (2016) Cas9-assisted recombineering in C. elegans: genome editing using in vivo assembly of linear DNAs. Nucleic Acids Res 44(15):e128
Auer TO, Del Bene F (2014) CRISPR/Cas9 and TALEN-mediated knock-in approaches in zebrafish. Methods 69(2):142–150
Liu Z, Chen O, Wall JBJ, Zheng M, Zhou Y, Wang L, Ruth Vaseghi H, Qian L, Liu J (2017) Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 7(1):2193
Chen X, Zaro JL, Shen WC (2013) Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 65(10):1357–1369
Koch B, Nijmeijer B, Kueblbeck M, Cai Y, Walther N, Ellenberg J (2018) Generation and validation of homozygous fluorescent knock-in cells using CRISPR-Cas9 genome editing. Nat Protoc 13(6):1465–1487
Kimple ME, Brill AL, Pasker RL (2013) Overview of affinity tags for protein purification. Curr Protoc Protein Sci 73:19–23
Rossolillo P, Kolesnikova O, Essabri K, Ramon Zamorano G, Poterszman A (in press) Production of multiprotein complexes using the Baculovirus expression system: homology based and Restriction Free cloning strategies for construct design. Methods Mol Biol
Anders C, Niewoehner O, Duerst A, Jinek M (2014) Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513(7519):569–573
Acknowledgments
We thank IGBMC cell culture and imaging facilities for assistance and fruitful discussions. 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 (R&D Project Funding), and Instruct-ULTRA as part of the European Union’s Horizon 2020 (Grant ID 731005) by Instruct-ERIC.
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Geny, S. et al. (2021). Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System and Double-Stranded DNA Donors. In: Poterszman, A. (eds) Multiprotein Complexes. Methods in Molecular Biology, vol 2247. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1126-5_3
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DOI: https://doi.org/10.1007/978-1-0716-1126-5_3
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