Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Yata 1111, Mishima, Shizuoka, 411-8540, Japan.
Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Yata 1111, Mishima, Shizuoka, 411-8540, Japan.
Nat Commun. 2020 Nov 11;11(1):5701. doi: 10.1038/s41467-020-19532-z.
Protein knockdown using the auxin-inducible degron (AID) technology is useful to study protein function in living cells because it induces rapid depletion, which makes it possible to observe an immediate phenotype. However, the current AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin. These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and to apply this method to mice. Here, we overcome these problems by taking advantage of a bump-and-hole approach to establish the AID version 2 (AID2) system. AID2, which employs an OsTIR1(F74G) mutant and a ligand, 5-Ph-IAA, shows no detectable leaky degradation, requires a 670-times lower ligand concentration, and achieves even quicker degradation than the conventional AID. We demonstrate successful generation of human cell mutants for genes that were previously difficult to deal with, and show that AID2 achieves rapid target depletion not only in yeast and mammalian cells, but also in mice.
利用生长素诱导的降解结构域（AID）技术进行蛋白敲低，可用于在活细胞中研究蛋白功能，因为它能诱导快速耗尽，从而可以观察到即时表型。然而，目前的 AID 系统有两个主要缺点：有泄漏性降解，并且需要高剂量的生长素。这些负面特征使得难以精确控制活细胞中感兴趣蛋白的表达水平，并且难以将该方法应用于小鼠。在这里，我们利用凹凸方法建立了 AID 版本 2（AID2）系统，克服了这些问题。AID2 采用 OsTIR1(F74G) 突变体和配体 5-Ph-IAA，没有检测到泄漏性降解，需要的配体浓度低 670 倍，降解速度甚至比传统的 AID 更快。我们成功地为以前难以处理的基因生成了人类细胞突变体，并表明 AID2 不仅在酵母和哺乳动物细胞中，而且在小鼠中也能实现快速靶蛋白耗尽。
