Chapter 3
Tagging Proteins with Fluorescent Reporters Using
the CRISPR/Cas9 System and Double-Stranded DNA Donors
Sylvain Geny, Simon Pichard, Alice Brion, Jean-Baptiste Renaud,
Sophie Jacquemin, Jean-Paul Concordet, and Arnaud Poterszman
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.
Key words CRISPR/Cas9, Genome editing, Double-stranded DNA donors, Fluorescent protein
1 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
39
Tagging Proteins with Fluorescent Reporters Using
the CRISPR/Cas9 System and Double-Stranded DNA Donors
Sylvain Geny, Simon Pichard, Alice Brion, Jean-Baptiste Renaud,
Sophie Jacquemin, Jean-Paul Concordet, and Arnaud Poterszman
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.
Key words CRISPR/Cas9, Genome editing, Double-stranded DNA donors, Fluorescent protein
1 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
39