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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...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9649518/ https://www.ncbi.nlm.nih.gov/pubmed/36386127 http://dx.doi.org/10.3389/fphar.2022.1015940 |
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author | Feng, Dongyan Zhong, Guorui Zuo, Qingxia Wan, Yanbin Xu, Wanqing He, Changsheng Lin, Cailing Huang, Dongchao Chen, Feng Huang, Lizhen |
author_facet | Feng, Dongyan Zhong, Guorui Zuo, Qingxia Wan, Yanbin Xu, Wanqing He, Changsheng Lin, Cailing Huang, Dongchao Chen, Feng Huang, Lizhen |
author_sort | Feng, Dongyan |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-9649518 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-96495182022-11-15 Knockout of ABC transporters by CRISPR/Cas9 contributes to reliable and accurate transporter substrate identification for drug discovery Feng, Dongyan Zhong, Guorui Zuo, Qingxia Wan, Yanbin Xu, Wanqing He, Changsheng Lin, Cailing Huang, Dongchao Chen, Feng Huang, Lizhen Front Pharmacol Pharmacology 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. Frontiers Media S.A. 2022-10-28 /pmc/articles/PMC9649518/ /pubmed/36386127 http://dx.doi.org/10.3389/fphar.2022.1015940 Text en Copyright © 2022 Feng, Zhong, Zuo, Wan, Xu, He, Lin, Huang, Chen and Huang. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Pharmacology Feng, Dongyan Zhong, Guorui Zuo, Qingxia Wan, Yanbin Xu, Wanqing He, Changsheng Lin, Cailing Huang, Dongchao Chen, Feng Huang, Lizhen Knockout of ABC transporters by CRISPR/Cas9 contributes to reliable and accurate transporter substrate identification for drug discovery |
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 | Pharmacology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9649518/ https://www.ncbi.nlm.nih.gov/pubmed/36386127 http://dx.doi.org/10.3389/fphar.2022.1015940 |
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