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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...

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Autores principales: Feng, Dongyan, Zhong, Guorui, Zuo, Qingxia, Wan, Yanbin, Xu, Wanqing, He, Changsheng, Lin, Cailing, Huang, Dongchao, Chen, Feng, Huang, Lizhen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
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.
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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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