Gene Tagging with the CRISPR-Cas9 System to Facilitate Macromolecular Complex Purification.
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, Poterszman A, Concordet JP
Lien Pubmed
Résumé
The need to generate modified cell lines that express tagged proteins of interest has become increasingly important. Here, we describe a detailed protocol for facile CRISPR/Cas9-mediated gene tagging and isolation of modified cells. In this protocol, we combine two previously published strategies that promote CRISPR/Cas9-mediated gene tagging: using chemically modified single-stranded oligonucleotides as donor templates and a co-selection strategy targeting the ATP1A1 gene at the same time as the gene of interest. Altogether, the protocol proposed here is both easier and saves time compared to other approaches for generating cells that express tagged proteins of interest, which is crucial to purify native complex from human cells.
Mots clés
CRISPR/Cas9, Co-selection, Complex purification, Single-stranded oligonucleotide donor
Référence
Methods Mol Biol. 2021 ;2305:153-174