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首页> 外文期刊>American Journal of Physiology >Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing
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Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing

机译：通过CRISPR / CAS介导的基因组编辑产生WNK1敲除细胞系

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Sodium-coupled SLC12 cation chloride cotransporters play important roles in cell volume and chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These cotransporters are phosphorylated and activated indirectly by With-No-Lysine (WNK) kinases through their downstream effector kinases, Ste20- and SPS1-related proline ala-nine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1). Multiple WNK kinases can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Deletion of specific WNKs from cells that natively express a functional WNK-SPAK/ OSR1 network will help resolve these knowledge gaps. Here, we outline a simple method to selectively knock out full-length WNK1 expression from mammalian cells using RNA-guided clustered regularly interspaced short palindromic repeats/Cas9 endonucleases. Two clonal cell lines were generated by using a single-guide RNA (sgRNA) targeting exon 1 of the WNK1 gene, which produced indels that abolished WNK1 protein expression. Both cell lines exhibited reduced endogenous WNK4 protein abundance, indicating that WNK1 is required for WNK4 stability. Consistent with an on-target effect, the reduced WNK4 abundance was associated with increased expression of the KLHL3/cullin-3 E3 ubiquitin ligase complex and was rescued by exogenous WNK1 overexpression. Although the morphology of the knockout cells was indistinguishable from control, they exhibited low baseline SPAK/OSR1 activity and failed to trigger regulatory volume increase after hypertonic stress, confirming an essential role for WNK1 in cell volume regulation. Collectively, our data show how this new, powerful, and accessible gene-editing technology can be used to dissect and analyze WNK signaling networks.
机译：钠偶联的SLC12阳离子氯化物分解商在细胞体积和氯化物稳态，上皮液分泌和肾小管盐重吸收中起重要作用。这些COTORANSPORTERS通过其下游效应激酶，STE20-和SPS1相关的脯氨酸ALA-富含富丙氨酸Ala-NINI-NINI激酶（SPAK）和氧化应激响应激酶1（OSR1）间接受赖氨酸（WNK）激酶磷酸化并间接地活化。多种WNK激酶可以在单个细胞类型内共存，尽管它们对SPAK / OSR1活化和盐传输的相对贡献保持不完全理解。删除自然表达功能性WNK-SPAK / OSR1网络的单元格中的特定WNK将有助于解决这些知识间隙。在这里，我们概述了一种简单的方法，可选择地使用RNA引导的聚类从哺乳动物细胞中选择性地敲出全长WNK1表达，所述RNA引导群体定期间隙短的短语重复/ CAS9内切核酸酶。通过使用WNK1基因的单引导RNA（SGRNA）产生两个克隆细胞系，其产生了废除WNK1蛋白表达的诱导。两种细胞系表现出降低的内源性WNK4蛋白质丰度，表明WNK1稳定性需要WNK1。与靶向效果一致，减少的WNK4丰度与KLHL3 / CULLIN-3 E3泛素连接酶复合物的表达增加有关，并通过外源WNK1过表达来抵抗。尽管敲除细胞的形态与对照无区别，但它们表现出低基线spak / OSR1活性，并且高渗应力后未能引发调节体积增加，确认WNK1在细胞体积调节中的基本作用。集体，我们的数据显示如何使用这种新的，强大和可访问的基因编辑技术来解剖和分析WNK信令网络。

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