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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.

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Figure 1: HPRT1 loss-of-function confers resistance to 6-thioguanine, as demonstrated by a forward genetic screen and CRISPR-mediated knockout.
Figure 2: Identification and validation of recessive triptolide-resistant ERCC3 alleles using CRISPR-mediated knock-in strategy.

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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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Yan Feng.

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Competing interests

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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