PT  - JOURNAL ARTICLE
AU  - Casey R. Dorr
AU  - Rory P. Remmel
AU  - Amutha Muthusamy
AU  - James Fisher
AU  - Branden S. Moriarity
AU  - Kazuto Yasuda
AU  - Baolin Wu
AU  - Weihua Guan
AU  - Erin G. Schuetz
AU  - William S. Oetting
AU  - Pamala A. Jacobson
AU  - Ajay K. Israni
TI  - CRISPR/Cas9 Genetic Modification of <em>CYP3A5 *3</em> in HuH-7 Human Hepatocyte Cell Line Leads to Cell Lines with Increased Midazolam and Tacrolimus Metabolism
AID  - 10.1124/dmd.117.076307
DP  - 2017 Aug 01
TA  - Drug Metabolism and Disposition
PG  - 957--965
VI  - 45
IP  - 8
4099  - http://dmd.aspetjournals.org/content/45/8/957.short
4100  - http://dmd.aspetjournals.org/content/45/8/957.full
SO  - Drug Metab Dispos2017 Aug 01; 45
AB  - 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.