Knockout of ABC transporters by CRISPR/Cas9 contributes to reliable and accurate transporter substrate identification for drug discovery.
Knockout of ABC transporters by CRISPR/Cas9 contributes to reliable and accurate transporter substrate identification for drug discovery.
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通过 CRISPR/Cas9 敲除 ABC 转运蛋白有助于药物发现中可靠、准确的转运蛋白底物识别
DOI：
10.3389/fphar.2022.1015940
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发表时间：
2022
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
--
作者：
在药物开发过程中，探索药物与转运体之间的关系至关重要。非人类MDCK或LLC-PK1细胞中的强背景信号和人类Caco-2细胞中抑制剂或RNAi的重叠干扰意味着敲除Caco-2细胞中的特定转运蛋白基因可能是一种理想的替代方法。然而，基因敲除（KO）技术在Caco-2细胞中的应用是具有挑战性的，因为通过长期的单克隆培养，获得具有双等位基因KO的快速生长的Caco-2亚克隆仍然效率低下。本研究利用低成本、高效、精准的基因编辑技术CRISPR/Cas9，对Caco-2细胞中的P-gp、BCRP和MRP2基因进行单敲除或双敲除。通过将其与单细胞扩增相结合，成功筛选和建立了快速生长的转运蛋白缺陷亚克隆。探针底物和三种蛋白酶抑制剂的双向运输实验表明，这些KO Caco-2模型可以很容易地获得更可靠和详细的数据。6个健壮的KO Caco-2亚克隆有助于有效的体外药物转运研究。
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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影响因子：
2
作者：
Fujimoto H;Higuchi M;Watanabe H;Koh Y;Ghosh AK;Mitsuya H;Tanoue N;Hamada A;Saito H
通讯作者：
Saito H
影响因子：
5.8
作者：
Agarwal, Sheetal;Pal, Dhananjay;Mitra, Ashim K.
通讯作者：
Mitra, Ashim K.
影响因子：
15.3
作者：
Ding, Xiaomeng;Hu, Xiaoyi;Yu, Qiang
通讯作者：
Yu, Qiang
影响因子：
3.6
作者：
Kopplow, K;Letschert, K;Keppler, D
通讯作者：
Keppler, D
影响因子：
6.1
作者：
Li, Size;Yu, Yiqun;Xiang, Xiaoqiang
通讯作者：
Xiang, Xiaoqiang