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Research ArticleArticles

CRISPR/Cas9 Genetic Modification of CYP3A5 *3 in HuH-7 Human Hepatocyte Cell Line Leads to Cell Lines with Increased Midazolam and Tacrolimus Metabolism

Casey R. Dorr, Rory P. Remmel, Amutha Muthusamy, James Fisher, Branden S. Moriarity, Kazuto Yasuda, Baolin Wu, Weihua Guan, Erin G. Schuetz, William S. Oetting, Pamala A. Jacobson and Ajay K. Israni
Drug Metabolism and Disposition August 2017, 45 (8) 957-965; DOI: https://doi.org/10.1124/dmd.117.076307
Casey R. Dorr
Molecular Epidemiology Laboratory, Minneapolis Medical Research Foundation, Minneapolis, Minnesota
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Rory P. Remmel
Molecular Epidemiology Laboratory, Minneapolis Medical Research Foundation, Minneapolis, Minnesota
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Amutha Muthusamy
Molecular Epidemiology Laboratory, Minneapolis Medical Research Foundation, Minneapolis, Minnesota
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James Fisher
Molecular Epidemiology Laboratory, Minneapolis Medical Research Foundation, Minneapolis, Minnesota
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Branden S. Moriarity
Molecular Epidemiology Laboratory, Minneapolis Medical Research Foundation, Minneapolis, Minnesota
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Kazuto Yasuda
Molecular Epidemiology Laboratory, Minneapolis Medical Research Foundation, Minneapolis, Minnesota
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Baolin Wu
Molecular Epidemiology Laboratory, Minneapolis Medical Research Foundation, Minneapolis, Minnesota
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Weihua Guan
Molecular Epidemiology Laboratory, Minneapolis Medical Research Foundation, Minneapolis, Minnesota
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Erin G. Schuetz
Molecular Epidemiology Laboratory, Minneapolis Medical Research Foundation, Minneapolis, Minnesota
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William S. Oetting
Molecular Epidemiology Laboratory, Minneapolis Medical Research Foundation, Minneapolis, Minnesota
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Pamala A. Jacobson
Molecular Epidemiology Laboratory, Minneapolis Medical Research Foundation, Minneapolis, Minnesota
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Ajay K. Israni
Molecular Epidemiology Laboratory, Minneapolis Medical Research Foundation, Minneapolis, Minnesota
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Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 engineering of the CYP3A5 *3 locus (rs776746) in human liver cell line HuH-7 (CYP3A5 *3/*3) has led to three CYP3A5 *1 cell lines by deletion of the exon 3B splice junction or point mutation. Cell lines CYP3A5 *1/*3 sd (single deletion), CYP3A5 *1/*1 dd (double deletion), or CYP3A5 *1/*3 pm (point mutation) expressed the CYP3A5 *1 mRNA and had elevated CYP3A5 mRNA (P < 0.0005 for all engineered cell lines) and protein expression compared with HuH-7. In metabolism assays, HuH-7 had less tacrolimus (all P < 0.05) or midazolam (MDZ) (all P < 0.005) disappearance than all engineered cell lines. HuH-7 had less 1-OH MDZ (all P < 0.0005) or 4-OH (all P < 0.005) production in metabolism assays than all bioengineered cell lines. We confirmed CYP3A5 metabolic activity with the CYP3A4 selective inhibitor CYP3CIDE. This is the first report of genomic CYP3A5 bioengineering in human cell lines with drug metabolism analysis.

Footnotes

    • Received April 13, 2017.
    • Accepted May 18, 2017.
  • This study was funded by a Minneapolis Medical Research Foundation Postdoctoral Career Development Award (to C.D.) and grant support from United States National institutes of Health National Institute for Allergy and Infectious Diseases [Grant AI U19 AI070119] (to A.I.).

  • https://doi.org/10.1124/dmd.117.076307.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

  • Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 45 (8)
Drug Metabolism and Disposition
Vol. 45, Issue 8
1 Aug 2017
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Research ArticleArticles

CRISPR Modification of CYP3A5 in HuH-7 Alters Drug Metabolism

Casey R. Dorr, Rory P. Remmel, Amutha Muthusamy, James Fisher, Branden S. Moriarity, Kazuto Yasuda, Baolin Wu, Weihua Guan, Erin G. Schuetz, William S. Oetting, Pamala A. Jacobson and Ajay K. Israni
Drug Metabolism and Disposition August 1, 2017, 45 (8) 957-965; DOI: https://doi.org/10.1124/dmd.117.076307

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Research ArticleArticles

CRISPR Modification of CYP3A5 in HuH-7 Alters Drug Metabolism

Casey R. Dorr, Rory P. Remmel, Amutha Muthusamy, James Fisher, Branden S. Moriarity, Kazuto Yasuda, Baolin Wu, Weihua Guan, Erin G. Schuetz, William S. Oetting, Pamala A. Jacobson and Ajay K. Israni
Drug Metabolism and Disposition August 1, 2017, 45 (8) 957-965; DOI: https://doi.org/10.1124/dmd.117.076307
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