A fast method for analyzing essential protein mutants in human cells.
Fiche publication
Date publication
février 2017
Journal
BioTechniques
Auteurs
Membres identifiés du Cancéropôle Est :
Pr CHATTON Bruno, Pr WEISS Etienne, Dr DONZEAU Mariel
Tous les auteurs :
Dietsch F, Donzeau M, Cordonnier AM, Weiss E, Chatton B, Vigneron M
Lien Pubmed
Résumé
Here we developed a complementation method for the study of essential genes in live human cells using the CRISPR/Cas9 system. Proteins encoded by essential genes were expressed using a derivative of the pCEP4 compensating plasmid in combination with Cas9 endonuclease targeting of the chromosomal genes. We show that this strategy can be applied to essential genes, such as those coding for proliferating cell nuclear antigen (PCNA) and DNA polymerase delta subunit 2 (POLD2). As demonstrated for the PCNA protein, our method allows mutational analysis of essential protein-coding sequences in live cells.
Mots clés
Cas9, human complementation assay, pCEP4
Référence
BioTechniques. 2017 Feb;62(2):80-82