AID2 technology gives superior degradation control

Protein knockdown by the auxin-inducible degron (AID) technology is useful to study protein function in living cells owing to its rapid depletion, that makes possible to observe an immediate phenotype. However, the current AID system has two major drawbacks; leaky degradation and use of a high dose of auxin. These negative features make difficult to precisely control the expression level of a protein of interest in living cells and to apply this method to mice. Here, we overcame these problems by taking an advantage of a bump-and-hole approach and established AID version 2 (AID2) systems. AID2 employing an OsTIR1(F74G) mutant and a new ligand, 5-Ph-IAA, show significantly low leaky degradation, requires a 670-fold less ligand concentration, and achieves even quicker degradation. We demonstrate that successful generation of human cell mutants for genes that were previously difficult to deal with, and that AID2 works not only in yeast and mammalian cells, but even in mice to achieve rapid target depletion.

利用生长素诱导降解系统（auxin-inducible degron, AID）技术实现蛋白质敲低，因其可快速降解靶蛋白并直接观测即时表型，在活细胞蛋白质功能研究中具备重要应用价值。然而，当前的AID系统存在两大核心弊端：渗漏性降解以及需使用高剂量生长素。这些缺陷使得难以在活细胞中精准调控目标蛋白的表达水平，同时也限制了该方法在小鼠模型中的应用。本研究借助“凹凸适配（bump-and-hole）”策略攻克了上述难题，成功构建了AID版本2（AID2）系统。采用OsTIR1(F74G)突变体与新型配体5-Ph-IAA的AID2系统，不仅渗漏性降解显著降低，所需配体浓度仅为原系统的1/670，且降解速度更为迅捷。本研究证实，AID2可成功构建此前难以操作的人类细胞基因突变异株，且该系统不仅可在酵母与哺乳动物细胞中发挥功能，甚至可应用于小鼠以实现靶蛋白的快速降解。