CRISPR/Cas9 Genetic Modification of CYP3A5 3 in HuH-7 Human Hepatocyte Cell Line Leads to Cell Lines with Increased Midazolam and Tacrolimus Metabolism
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 d...
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| Veröffentlicht in: | Drug metabolism and disposition 2017-08, Vol.45 (8), p.957-965 |
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| creator | Dorr, Casey R. Remmel, Rory P. Muthusamy, Amutha Fisher, James Moriarity, Branden S. Yasuda, Kazuto Wu, Baolin Guan, Weihua Schuetz, Erin G. Oetting, William S. Jacobson, Pamala A. Israni, Ajay K. |
| description | 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. |
| doi_str_mv | 10.1124/dmd.117.076307 |
| format | Article |
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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.</description><identifier>ISSN: 0090-9556</identifier><identifier>EISSN: 1521-009X</identifier><identifier>DOI: 10.1124/dmd.117.076307</identifier><identifier>PMID: 28533324</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Bioengineering ; Biotechnology ; Cell Line ; Cell lines ; Clonal deletion ; Clustered Regularly Interspaced Short Palindromic Repeats - genetics ; CRISPR ; CRISPR-Cas Systems - genetics ; Cytochrome P-450 CYP3A - genetics ; Drug metabolism ; Gene deletion ; Gene expression ; Genetic modification ; Hepatocytes ; Hepatocytes - metabolism ; Humans ; Liver ; Liver - metabolism ; Male ; Metabolism ; Midazolam ; Midazolam - metabolism ; Middle Aged ; mRNA ; Mutation ; Point mutation ; Point Mutation - genetics ; Protein turnover ; RNA, Messenger - genetics ; Sequence Deletion - genetics ; Tacrolimus ; Tacrolimus - metabolism</subject><ispartof>Drug metabolism and disposition, 2017-08, Vol.45 (8), p.957-965</ispartof><rights>2017 American Society for Pharmacology and Experimental Therapeutics</rights><rights>Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.</rights><rights>Copyright Lippincott Williams & Wilkins Ovid Technologies Aug 1, 2017</rights><rights>Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics 2017</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3827-4b5af028f6d1a4203bf783f3505781c80ba1ada067f2b9d38abfb78bec44eabe3</citedby><cites>FETCH-LOGICAL-c3827-4b5af028f6d1a4203bf783f3505781c80ba1ada067f2b9d38abfb78bec44eabe3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,780,784,885,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28533324$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Dorr, Casey R.</creatorcontrib><creatorcontrib>Remmel, Rory P.</creatorcontrib><creatorcontrib>Muthusamy, Amutha</creatorcontrib><creatorcontrib>Fisher, James</creatorcontrib><creatorcontrib>Moriarity, Branden S.</creatorcontrib><creatorcontrib>Yasuda, Kazuto</creatorcontrib><creatorcontrib>Wu, Baolin</creatorcontrib><creatorcontrib>Guan, Weihua</creatorcontrib><creatorcontrib>Schuetz, Erin G.</creatorcontrib><creatorcontrib>Oetting, William S.</creatorcontrib><creatorcontrib>Jacobson, Pamala A.</creatorcontrib><creatorcontrib>Israni, Ajay K.</creatorcontrib><title>CRISPR/Cas9 Genetic Modification of CYP3A5 3 in HuH-7 Human Hepatocyte Cell Line Leads to Cell Lines with Increased Midazolam and Tacrolimus Metabolism</title><title>Drug metabolism and disposition</title><addtitle>Drug Metab Dispos</addtitle><description>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.</description><subject>Bioengineering</subject><subject>Biotechnology</subject><subject>Cell Line</subject><subject>Cell lines</subject><subject>Clonal deletion</subject><subject>Clustered Regularly Interspaced Short Palindromic Repeats - genetics</subject><subject>CRISPR</subject><subject>CRISPR-Cas Systems - genetics</subject><subject>Cytochrome P-450 CYP3A - genetics</subject><subject>Drug metabolism</subject><subject>Gene deletion</subject><subject>Gene expression</subject><subject>Genetic modification</subject><subject>Hepatocytes</subject><subject>Hepatocytes - metabolism</subject><subject>Humans</subject><subject>Liver</subject><subject>Liver - metabolism</subject><subject>Male</subject><subject>Metabolism</subject><subject>Midazolam</subject><subject>Midazolam - metabolism</subject><subject>Middle Aged</subject><subject>mRNA</subject><subject>Mutation</subject><subject>Point mutation</subject><subject>Point Mutation - genetics</subject><subject>Protein turnover</subject><subject>RNA, Messenger - genetics</subject><subject>Sequence Deletion - genetics</subject><subject>Tacrolimus</subject><subject>Tacrolimus - metabolism</subject><issn>0090-9556</issn><issn>1521-009X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1UcFu1DAQjRCIbgtXjsgS52zt2F47F6Qqgu5Ku6IqRYKTNbYn1FUSL3FSVH6kv4vRlgIHLp4nz_Ob53lF8YrRJWOVOPW9z0AtqVpxqp4UCyYrVlJaf35aLHKhZS3l6qg4TumGUiYEr58XR5WWnPNKLIr75nLz8eLytIFUk3MccAqO7KIPbXAwhTiQ2JLmywU_k4STMJD1vC5VPnvIGPcwRXc3IWmw68g2DEi2CD6RKf65SuR7mK7JZnAjQkJPdsHDj9hBT2Dw5ArcGLvQz4nscAKbcepfFM9a6BK-fKgnxaf3766adbn9cL5pzral47pSpbASWlrpduUZiIpy2yrNWy6pVJo5TS0w8EBXqq1s7bkG21qlLTohECzyk-LtQXc_2x69w2EaoTP7MfQw3pkIwfzbGcK1-RpvjZRMK6azwJsHgTF-mzFN5ibO45A9myobqwUTssqs5YGVv5rSiO3jBEbNryBNDjIDZQ5B5gev__b1SP-dXCboAwHzdm4Djia5gINDH0Z0k_Ex_E_7J-srrKc</recordid><startdate>201708</startdate><enddate>201708</enddate><creator>Dorr, Casey R.</creator><creator>Remmel, Rory P.</creator><creator>Muthusamy, Amutha</creator><creator>Fisher, James</creator><creator>Moriarity, Branden S.</creator><creator>Yasuda, Kazuto</creator><creator>Wu, Baolin</creator><creator>Guan, Weihua</creator><creator>Schuetz, Erin G.</creator><creator>Oetting, William S.</creator><creator>Jacobson, Pamala A.</creator><creator>Israni, Ajay K.</creator><general>Elsevier Inc</general><general>American Society for Pharmacology and Experimental Therapeutics, Inc</general><general>The American Society for Pharmacology and Experimental Therapeutics</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QO</scope><scope>7TK</scope><scope>7U7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>5PM</scope></search><sort><creationdate>201708</creationdate><title>CRISPR/Cas9 Genetic Modification of CYP3A5 3 in HuH-7 Human Hepatocyte Cell Line Leads to Cell Lines with Increased Midazolam and Tacrolimus Metabolism</title><author>Dorr, Casey R. ; Remmel, Rory P. ; Muthusamy, Amutha ; Fisher, James ; Moriarity, Branden S. ; Yasuda, Kazuto ; Wu, Baolin ; Guan, Weihua ; Schuetz, Erin G. ; Oetting, William S. ; Jacobson, Pamala A. ; Israni, Ajay K.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3827-4b5af028f6d1a4203bf783f3505781c80ba1ada067f2b9d38abfb78bec44eabe3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Bioengineering</topic><topic>Biotechnology</topic><topic>Cell Line</topic><topic>Cell lines</topic><topic>Clonal deletion</topic><topic>Clustered Regularly Interspaced Short Palindromic Repeats - genetics</topic><topic>CRISPR</topic><topic>CRISPR-Cas Systems - genetics</topic><topic>Cytochrome P-450 CYP3A - genetics</topic><topic>Drug metabolism</topic><topic>Gene deletion</topic><topic>Gene expression</topic><topic>Genetic modification</topic><topic>Hepatocytes</topic><topic>Hepatocytes - metabolism</topic><topic>Humans</topic><topic>Liver</topic><topic>Liver - metabolism</topic><topic>Male</topic><topic>Metabolism</topic><topic>Midazolam</topic><topic>Midazolam - metabolism</topic><topic>Middle Aged</topic><topic>mRNA</topic><topic>Mutation</topic><topic>Point mutation</topic><topic>Point Mutation - genetics</topic><topic>Protein turnover</topic><topic>RNA, Messenger - genetics</topic><topic>Sequence Deletion - genetics</topic><topic>Tacrolimus</topic><topic>Tacrolimus - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Dorr, Casey R.</creatorcontrib><creatorcontrib>Remmel, Rory P.</creatorcontrib><creatorcontrib>Muthusamy, Amutha</creatorcontrib><creatorcontrib>Fisher, James</creatorcontrib><creatorcontrib>Moriarity, Branden S.</creatorcontrib><creatorcontrib>Yasuda, Kazuto</creatorcontrib><creatorcontrib>Wu, Baolin</creatorcontrib><creatorcontrib>Guan, Weihua</creatorcontrib><creatorcontrib>Schuetz, Erin G.</creatorcontrib><creatorcontrib>Oetting, William S.</creatorcontrib><creatorcontrib>Jacobson, Pamala A.</creatorcontrib><creatorcontrib>Israni, Ajay K.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Toxicology Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Drug metabolism and disposition</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Dorr, Casey R.</au><au>Remmel, Rory P.</au><au>Muthusamy, Amutha</au><au>Fisher, James</au><au>Moriarity, Branden S.</au><au>Yasuda, Kazuto</au><au>Wu, Baolin</au><au>Guan, Weihua</au><au>Schuetz, Erin G.</au><au>Oetting, William S.</au><au>Jacobson, Pamala A.</au><au>Israni, Ajay K.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>CRISPR/Cas9 Genetic Modification of CYP3A5 3 in HuH-7 Human Hepatocyte Cell Line Leads to Cell Lines with Increased Midazolam and Tacrolimus Metabolism</atitle><jtitle>Drug metabolism and disposition</jtitle><addtitle>Drug Metab Dispos</addtitle><date>2017-08</date><risdate>2017</risdate><volume>45</volume><issue>8</issue><spage>957</spage><epage>965</epage><pages>957-965</pages><issn>0090-9556</issn><eissn>1521-009X</eissn><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.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>28533324</pmid><doi>10.1124/dmd.117.076307</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Bioengineering Biotechnology Cell Line Cell lines Clonal deletion Clustered Regularly Interspaced Short Palindromic Repeats - genetics CRISPR CRISPR-Cas Systems - genetics Cytochrome P-450 CYP3A - genetics Drug metabolism Gene deletion Gene expression Genetic modification Hepatocytes Hepatocytes - metabolism Humans Liver Liver - metabolism Male Metabolism Midazolam Midazolam - metabolism Middle Aged mRNA Mutation Point mutation Point Mutation - genetics Protein turnover RNA, Messenger - genetics Sequence Deletion - genetics Tacrolimus Tacrolimus - metabolism |
| title | CRISPR/Cas9 Genetic Modification of CYP3A5 3 in HuH-7 Human Hepatocyte Cell Line Leads to Cell Lines with Increased Midazolam and Tacrolimus Metabolism |
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