DNA sequencing and CRISPR-Cas9 gene editing for target validation in mammalian cells

June 15th, 2014 by Yegor Smurnyy

Nature Chemical Biology 10, 623 (2014). doi:10.1038/nchembio.1550

Authors: Yegor Smurnyy, Mi Cai, Hua Wu, Elizabeth McWhinnie, John A Tallarico, Yi Yang & Yan Feng

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.

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