title: A fast method for analyzing essential protein mutants in human cells.
abstract: 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.
authors.affiliation: Université de Strasbourg, Biotechnologie et Signalisation Cellulaire, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France.
authors.forename: Frank
authors.initials: F
authors.lastname: Dietsch
authors.affiliation: Université de Strasbourg, Biotechnologie et Signalisation Cellulaire, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France.
authors.forename: Mariel
authors.initials: M
authors.lastname: Donzeau
authors.affiliation: Université de Strasbourg, Biotechnologie et Signalisation Cellulaire, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France.
authors.forename: Agnes M
authors.initials: AM
authors.lastname: Cordonnier
authors.affiliation: Université de Strasbourg, Biotechnologie et Signalisation Cellulaire, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France.
authors.forename: Etienne
authors.initials: E
authors.lastname: Weiss
authors.affiliation: Université de Strasbourg, Biotechnologie et Signalisation Cellulaire, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Chatton
authors.affiliation: Université de Strasbourg, Biotechnologie et Signalisation Cellulaire, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Vigneron
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D003584:Cytological Techniques; D004252:DNA Mutational Analysis / Q000379:methods*; D055786:Gene Knockout Techniques; D020043:Genes, Essential / Q000235:genetics*; D005816:Genetic Complementation Test / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics
chemical_list: D018809:Proliferating Cell Nuclear Antigen
keywords: Cas9; human complementation assay; pCEP4