Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors
Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors
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DOI：
10.1016/j.celrep.2016.03.001
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发表时间：
2016-04-05
期刊：
影响因子：
8.8
通讯作者：
Kanemaki, Masato T.
中科院分区：
文献类型：
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作者：
Natsume, Toyoaki;Kiyomitsu, Tomomi;Kanemaki, Masato T.
研究必需蛋白质的作用取决于快速失活的方法，以便研究直接的表型后果。生长素诱导降解决定子 (AID) 技术可以快速消耗动物细胞和真菌中的蛋白质，但其在人类细胞中的应用因标记内源蛋白质的困难而受到限制。我们开发了一种简单且可扩展的基于 CRISPR/Cas 的方法，通过使用具有合成短同源臂的供体结构来标记人 HCT116 和小鼠胚胎干 (ES) 细胞中的内源蛋白。通过将 AID 标记与 CRISPR/Cas 相结合，我们在 HCT116 细胞中生成了必需的核蛋白和细胞质蛋白的条件等位基因，这些等位基因在向培养基中添加生长素后可以非常迅速地耗尽。这种方法应该极大地促进必需蛋白质的功能分析，特别是那些以前未知功能的蛋白质。
Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES) cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function.