Conventional screening methods for deubiquitinating enzymes (DUBs) have important limitations. A loss-of-function study based on the knockout of DUB genes in mammalian cells can provide an excellent model for exploring DUB function. Here, we used CRISPR-Cas9 to perform genome-scale knockout of the entire set of genes encoding ubiquitin-specific proteases (USPs), a DUB subfamily, and then systematically screened for DUBs that stabilize the Cdc25A oncoprotein. USP3 was identified as a deubiquitinase of Cdc25A. USP3 depletion reduces the Cdc25A protein level, resulting in a significant delay in cell-cycle progression, and reduces the growth of cervical tumor xenografts in nude mice. Clinically, USP3 expression is positively correlated with Cdc25A protein expression and the poorest survival in breast cancer. We envision that our DUB knockout library kit will facilitate genome-scale screening of functional DUBs for target proteins of interest in a wide range of biomedical fields.

中文翻译：

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去泛素化酶亚家族的基因组规模筛选将 USP3 鉴定为 Cdc25A 调节癌症细胞周期的稳定剂。

去泛素化酶 (DUB) 的常规筛选方法具有重要的局限性。基于哺乳动物细胞中 DUB 基因敲除的功能丧失研究可为探索 DUB 功能提供极好的模型。在这里，我们使用 CRISPR-Cas9 对整个编码泛素特异性蛋白酶 (USP) 的基因组进行基因组规模敲除，这是一个 DUB 亚家族，然后系统地筛选了稳定 Cdc25A 癌蛋白的 DUB。USP3 被鉴定为 Cdc25A 的去泛素化酶。USP3 消耗降低了 Cdc25A 蛋白水平，导致细胞周期进程显着延迟，并减少了裸鼠宫颈肿瘤异种移植物的生长。临床上，USP3 表达与 Cdc25A 蛋白表达和乳腺癌中最差的存活率呈正相关。