Creating Knockouts of Conserved Oligomeric Golgi Complex Subunits Using CRISPR-Mediated Gene Editing Paired with a Selection Strategy Based on Glycosylation Defects Associated with Impaired COG Complex Function.
Creating Knockouts of Conserved Oligomeric Golgi Complex Subunits Using CRISPR-Mediated Gene Editing Paired with a Selection Strategy Based on Glycosylation Defects Associated with Impaired COG Complex Function.
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DOI：
10.1007/978-1-4939-6463-5_12
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Lupashin VV
中科院分区：
文献类型：
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作者：
Blackburn JB;Lupashin VV
保守寡聚体高尔基体（COG）复合物是一个关键的进化保守的多亚基蛋白质机制，调节高尔基体内运输囊泡的束缚和融合。高尔基体特异性地促进糖缀合物的分选和复杂糖基化。如果没有适当的糖基化和加工，蛋白质和脂质将被错误定位和/或功能受损。高尔基体糖基化机制通过顺行和逆行运输的仔细平衡保持稳态，以确保糖基化酶及其底物的适当定位。与膜运输的其他步骤一样，这种平衡由囊泡运输机制维持，所述囊泡运输机制包括COPI囊泡外壳蛋白、SNARE、Rabs以及卷曲螺旋和多亚基囊泡系链。COG复合物与其他膜运输组分相互作用，并且对于高尔基体糖基化机器的正确定位是必不可少的。在这里，我们描述了使用CRISPR介导的基因编辑结合与COG复合物在糖基化稳态中的作用直接相关的基于表型的选择策略来获得COG复合物亚基敲除（科斯）。这导致了HEK 293 T细胞中每个COG亚基的克隆科斯，并提供了进一步探测COG复合物在高尔基体稳态中的作用的能力。
The Conserved Oligomeric Golgi (COG) complex is a key evolutionally conserved multisubunit protein machinery that regulates tethering and fusion of intra-Golgi transport vesicles. The Golgi apparatus specifically promotes sorting and complex glycosylation of glycoconjugates. Without proper glycosylation and processing, proteins and lipids will be mislocalized and/or have impaired function. The Golgi glycosylation machinery is kept in homeostasis by a careful balance of anterograde and retrograde trafficking to ensure proper localization of the glycosylation enzymes and their substrates. This balance, like other steps of membrane trafficking, is maintained by vesicle trafficking machinery that includes COPI vesicular coat proteins, SNAREs, Rabs, and both coiled-coil and multi-subunit vesicular tethers. COG complex interacts with other membrane trafficking components and is essential for proper localization of Golgi glycosylation machinery. Here we describe using CRISPR-mediated gene editing coupled with a phenotype-based selection strategy directly linked to the COG complex’s role in glycosylation homeostasis to obtain COG complex subunit knock-outs (KOs). This has resulted in clonal KOs for each COG subunit in HEK293T cells and gives the ability to further probe the role of the COG complex in Golgi homeostasis.