title: DNA sequencing and CRISPR-Cas9 gene editing for target validation in mammalian cells.
abstract: Identification and validation of drug-resistant mutations can provide important insights into the mechanism of action of a compound. Here we demonstrate the feasibility of such an approach in mammalian cells using next-generation sequencing of drug-resistant clones and CRISPR-Cas9-mediated gene editing on two drug-target pairs, 6-thioguanine-HPRT1 and triptolide-ERCC3. We showed that disrupting functional HPRT1 allele or introducing ERCC3 point mutations by gene editing can confer drug resistance in cells.
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Yegor
authors.initials: Y
authors.lastname: Smurnyy
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Mi
authors.initials: M
authors.lastname: Cai
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Hua
authors.initials: H
authors.lastname: Wu
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: McWhinnie
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Tallarico
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Feng
mesh_terms: D000818:Animals; D002460:Cell Line / Q000187:drug effects; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004265:DNA Helicases / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D004224:Diterpenes / Q000494:pharmacology; D004351:Drug Resistance / Q000187:drug effects / Q000235:genetics; D004720:Endonucleases / Q000235:genetics*; D004852:Epoxy Compounds / Q000494:pharmacology; D045325:HCT116 Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008322:Mammals; D010616:Phenanthrenes / Q000494:pharmacology; D017354:Point Mutation; D015203:Reproducibility of Results; D013866:Thioguanine / Q000494:pharmacology
chemical_list: D004268:DNA-Binding Proteins; D004224:Diterpenes; D004852:Epoxy Compounds; D010616:Phenanthrenes; C072022:XPBC-ERCC-3 protein; C001899:triptolide; D007041:Hypoxanthine Phosphoribosyltransferase; D004720:Endonucleases; D004265:DNA Helicases; D013866:Thioguanine