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.
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Acknowledgements
The authors would like to thank N. Guo for technical help on CRISPR-Cas9 cloning. Y.S. and M.C. would like to thank the Novartis Postdoc Office for support during their training.
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Y.F., Y.Y., J.A.T. and Y.S. conceived the study, Y.S., M.C., H.W. and E.M. conducted experiments, Y.S. performed bioinformatic analysis, and Y.S. and Y.F. wrote the manuscript with additional help from all authors.
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At the time the research was performed, the authors were employees of Novartis, and the research was fully funded by Novartis.
Supplementary information
Supplementary Text and Figures
Supplementary Results, Supplementary Tables 1–14 and Supplementary Figures 1–11. (PDF 1443 kb)
Supplementary Data Set 1
Results of next generation exome sequencing of Triptolide resistant KBM7 clones (XLSX 36 kb)
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Smurnyy, Y., Cai, M., Wu, H. et al. DNA sequencing and CRISPR-Cas9 gene editing for target validation in mammalian cells. Nat Chem Biol 10, 623–625 (2014). https://doi.org/10.1038/nchembio.1550
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DOI: https://doi.org/10.1038/nchembio.1550
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