Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System and Double-Stranded DNA Donors.
Methods Mol Biol
; 2247: 39-57, 2021.
Article
en En
| MEDLINE
| ID: mdl-33301111
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Proteínas Recombinantes de Fusión
/
ADN
/
Proteínas Fluorescentes Verdes
/
Sistemas CRISPR-Cas
/
Edición Génica
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2021
Tipo del documento:
Article
País de afiliación:
Francia