Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System and Double-Stranded DNA Donors
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 d
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Introduction Molecular complexes of interacting proteins govern virtually all biological processes such as metabolism, cell signaling, DNA repair, and gene expression. Macromolecular assemblies are also of great biomedical relevance as their dysfunctions underlie a number of diseases, and deliberate inhibition of protein–protein interactions is an increasingly common strategy in drug discovery [1–3]. To fully understand their biological roles, it is essential to study the structure and function of intact protein assemblies. Although advanced recombinant protein technologies are available to reconstitute multiprotein complexes composed of ten or more subunits, many protein complexes are difficult to obtain using recombinant methods. An additional hurdle is that the subunit composition of complexes is not always known well enough to proceed to
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247, https://doi.org/10.1007/978-1-0716-1126-5_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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Sylvain Geny et al.
reconstitution of functional entities and therefore to structure determination. The CRISPR/Cas9 system has revolutionized many fields of life sciences by making it possible to modify the genome sequence with unprecedented efficiency and precision [4, 5]. For example, it is now possible to insert fusion tags at endogenous loci of mammalian cells, providing an efficient way to undertake affinity purification of macromolecular complexes and/or visualize their distribution and dynamics in a cellular context [6, 7]. We have recently detailed a simplified protocol for CRISPR/Cas9-mediated gene tagging in human cell lines using chemically modified singlestranded oligonucleotides encoding a small affinity tag as donor template and a co-selection strategy targeting the ATP1A1 gene [8] (to be published in MiB, Editor R Owens). Here we describe a procedure based on the use of the CRISPR/Cas9 system and double-stranded DNA donors for tagging the protein of interest with a fluorescent reporter and detail the generation of an U2-OS:: XPB-GFP knocked-in cell line expressing a XPB-GFP fusion protein. XPB is a subunit of the TFIIH complex, essential in initiation of DNA transcription by RNA polymerase II and DNA repair by nucleotide excision repair [9, 10]. This XPB-GFP-tagged U2-OS cell line was instrumental to establish a partnership between TFIIH and the histone acetyl transferase GCN5 and the impact of TFIIH on GCN5 activity with important consequences on gene expression and chromatin structure [11].
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Materials Procedures described here need access to standard equipment for molecular biology (PCR amplification, agarose gel analysis, bacteria transformation, access to a DNA sequencing/synthesis service, etc.), cell culture (cryo-container and liquid nitrogen source, temperature- and CO2-controlled incubator, laminar flow hood, centrifuge with adaptor for 15 and 50 mL tubes, cell counter), cell microscopy (fluorescence microscope with
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