As a potent and convenient genome-editing tool, Cas9 has been widely used in biomedical research and evaluated in treating human diseases. Numerous engineered variants of Cas9, dCas9 and other related prokaryotic endonucleases have been identified. However, as these bacterial enzymes are not naturally present in mammalian cells, whether and how bacterial Cas9 proteins are recognized and regulated by mammalian hosts remain poorly understood. Here, we identify Keap1 as a mammalian endogenous E3 ligase that targets Cas9/dCas9/Fanzor for ubiquitination and degradation in an ‘ETGE’-like degron-dependent manner. Cas9-‘ETGE’-like degron mutants evading Keap1 recognition display enhanced gene editing ability in cells. dCas9-‘ETGE’-like degron mutants exert extended protein half-life and protein retention on chromatin, leading to improved CRISPRa and CRISPRi efficacy. Moreover, Cas9 binding to Keap1 also impairs Keap1 function by competing with Keap1 substrates or binding partners for Keap1 binding, while engineered Cas9 mutants show less perturbation of Keap1 biology. Thus, our study reveals a mammalian specific Cas9 regulation and provides new Cas9 designs not only with enhanced gene regulatory capacity but also with minimal effects on disrupting endogenous Keap1 signaling.

中文翻译：

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工程化的 Cas9 变体绕过了 Keap1 介导的人类细胞降解，提高了表观基因组编辑效率


Cas9 作为一种有效且方便的基因组编辑工具，已广泛应用于生物医学研究，并在治疗人类疾病方面得到评价。已鉴定出 Cas9、dCas9 和其他相关原核核酸内切酶的许多工程变体。然而，由于这些细菌酶并非天然存在于哺乳动物细胞中，因此哺乳动物宿主是否以及如何识别和调节细菌 Cas9 蛋白仍然知之甚少。在这里，我们将 Keap1 鉴定为哺乳动物内源性 E3 连接酶，它以“ETGE”样 degron 依赖性方式靶向 Cas9/dCas9/Fanzor 进行泛素化和降解。逃避 Keap1 识别的 Cas9-'ETGE' 样 degron 突变体在细胞中显示出增强的基因编辑能力。dCas9-'ETGE' 样 degron 突变体延长蛋白质半衰期和蛋白质保留对染色质的影响，从而提高 CRISPRa 和 CRISPRi 功效。此外，Cas9 与 Keap1 的结合还通过与 Keap1 底物或结合伴侣竞争 Keap1 结合来损害 Keap1 功能，而工程化的 Cas9 突变体对 Keap1 生物学的扰动较小。因此，我们的研究揭示了哺乳动物特异性的 Cas9 调控，并提供了新的 Cas9 设计，不仅具有增强的基因调控能力，而且对破坏内源性 Keap1 信号传导的影响最小。