Knockout of ABC transporters by CRISPR/Cas9 contributes to reliable and accurate transporter substrate identification for drug discovery
It is essential to explore the relationship between drugs and transporters in the process of drug development. Strong background signals in nonhuman MDCK or LLC-PK1 cells and overlapping interference of inhibitors or RNAi in human Caco-2 cells mean that an ideal alternative could be to knock out spe...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-10-01
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| Series: | Frontiers in Pharmacology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fphar.2022.1015940/full |
| _version_ | 1811257367543676928 |
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| author | Dongyan Feng Guorui Zhong Qingxia Zuo Yanbin Wan Wanqing Xu Changsheng He Cailing Lin Dongchao Huang Feng Chen Lizhen Huang |
| author_facet | Dongyan Feng Guorui Zhong Qingxia Zuo Yanbin Wan Wanqing Xu Changsheng He Cailing Lin Dongchao Huang Feng Chen Lizhen Huang |
| author_sort | Dongyan Feng |
| collection | DOAJ |
| description | It is essential to explore the relationship between drugs and transporters in the process of drug development. Strong background signals in nonhuman MDCK or LLC-PK1 cells and overlapping interference of inhibitors or RNAi in human Caco-2 cells mean that an ideal alternative could be to knock out specific transporter genes in Caco-2 cells. However, the application of gene knockout (KO) to Caco-2 cells is challenging because it is still inefficient to obtain rapidly growing Caco-2 subclones with double-allele KO through long-term monoclonal cultivation. Herein, CRISPR/Cas9, a low cost but more efficient and precise gene editing technology, was utilized to singly or doubly knockout the P-gp, BCRP, and MRP2 genes in Caco-2 cells. By combining this with single cell expansion, rapidly growing transporter-deficient subclones were successfully screened and established. Bidirectional transport assays with probe substrates and three protease inhibitors indicated that more reliable and detailed data could be drawn easily with these KO Caco-2 models. The six robust KO Caco-2 subclones could contribute to efficient in vitro drug transport research. |
| first_indexed | 2024-04-12T17:56:20Z |
| format | Article |
| id | doaj.art-1e42dbdb7477402aad696ad97dc92c5e |
| institution | Directory Open Access Journal |
| issn | 1663-9812 |
| language | English |
| last_indexed | 2024-04-12T17:56:20Z |
| publishDate | 2022-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Pharmacology |
| spelling | doaj.art-1e42dbdb7477402aad696ad97dc92c5e2022-12-22T03:22:20ZengFrontiers Media S.A.Frontiers in Pharmacology1663-98122022-10-011310.3389/fphar.2022.10159401015940Knockout of ABC transporters by CRISPR/Cas9 contributes to reliable and accurate transporter substrate identification for drug discoveryDongyan Feng0Guorui Zhong1Qingxia Zuo2Yanbin Wan3Wanqing Xu4Changsheng He5Cailing Lin6Dongchao Huang7Feng Chen8Lizhen Huang9School of Biology and Biological Engineering, South China University of Technology, Guangzhou, ChinaBioinformatics Institute, Agency for Science, Technology, and Research (A*STAR), Singapore, SingaporeSchool of Biology and Biological Engineering, South China University of Technology, Guangzhou, ChinaSchool of Biology and Biological Engineering, South China University of Technology, Guangzhou, ChinaSchool of Biology and Biological Engineering, South China University of Technology, Guangzhou, ChinaSchool of Biology and Biological Engineering, South China University of Technology, Guangzhou, ChinaSchool of Biology and Biological Engineering, South China University of Technology, Guangzhou, ChinaSchool of Biology and Biological Engineering, South China University of Technology, Guangzhou, ChinaSchool of Biology and Biological Engineering, South China University of Technology, Guangzhou, ChinaSchool of Biology and Biological Engineering, South China University of Technology, Guangzhou, ChinaIt is essential to explore the relationship between drugs and transporters in the process of drug development. Strong background signals in nonhuman MDCK or LLC-PK1 cells and overlapping interference of inhibitors or RNAi in human Caco-2 cells mean that an ideal alternative could be to knock out specific transporter genes in Caco-2 cells. However, the application of gene knockout (KO) to Caco-2 cells is challenging because it is still inefficient to obtain rapidly growing Caco-2 subclones with double-allele KO through long-term monoclonal cultivation. Herein, CRISPR/Cas9, a low cost but more efficient and precise gene editing technology, was utilized to singly or doubly knockout the P-gp, BCRP, and MRP2 genes in Caco-2 cells. By combining this with single cell expansion, rapidly growing transporter-deficient subclones were successfully screened and established. Bidirectional transport assays with probe substrates and three protease inhibitors indicated that more reliable and detailed data could be drawn easily with these KO Caco-2 models. The six robust KO Caco-2 subclones could contribute to efficient in vitro drug transport research.https://www.frontiersin.org/articles/10.3389/fphar.2022.1015940/fulltransporter substrate identificationABC efflux transportersCRISPR/Cas9gene knockoutCaco-2 |
| spellingShingle | Dongyan Feng Guorui Zhong Qingxia Zuo Yanbin Wan Wanqing Xu Changsheng He Cailing Lin Dongchao Huang Feng Chen Lizhen Huang Knockout of ABC transporters by CRISPR/Cas9 contributes to reliable and accurate transporter substrate identification for drug discovery Frontiers in Pharmacology transporter substrate identification ABC efflux transporters CRISPR/Cas9 gene knockout Caco-2 |
| title | Knockout of ABC transporters by CRISPR/Cas9 contributes to reliable and accurate transporter substrate identification for drug discovery |
| title_full | Knockout of ABC transporters by CRISPR/Cas9 contributes to reliable and accurate transporter substrate identification for drug discovery |
| title_fullStr | Knockout of ABC transporters by CRISPR/Cas9 contributes to reliable and accurate transporter substrate identification for drug discovery |
| title_full_unstemmed | Knockout of ABC transporters by CRISPR/Cas9 contributes to reliable and accurate transporter substrate identification for drug discovery |
| title_short | Knockout of ABC transporters by CRISPR/Cas9 contributes to reliable and accurate transporter substrate identification for drug discovery |
| title_sort | knockout of abc transporters by crispr cas9 contributes to reliable and accurate transporter substrate identification for drug discovery |
| topic | transporter substrate identification ABC efflux transporters CRISPR/Cas9 gene knockout Caco-2 |
| url | https://www.frontiersin.org/articles/10.3389/fphar.2022.1015940/full |
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