Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System and Double-Stranded DNA Donors.
Fiche publication
Date publication
janvier 2021
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr POTERSZMAN Arnaud
Tous les auteurs :
Geny S, Pichard S, Brion A, Renaud JB, Jacquemin S, Concordet JP, Poterszman A
Lien Pubmed
Résumé
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.
Mots clés
CRISPR/Cas9, Double-stranded DNA donors, Fluorescent protein, Genome editing
Référence
Methods Mol Biol. 2021 ;2247:39-57