Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Department of Biochemistry and Biophysics, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Nucleic Acids Res. 2024 Oct 28;52(19):11536-11551. doi: 10.1093/nar/gkae761.
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.
作为一种强大且便捷的基因组编辑工具，Cas9 已被广泛应用于生物医学研究，并在人类疾病的治疗中得到了评估。大量经过工程改造的 Cas9、dCas9 和其他相关的原核内切酶已被鉴定出来。然而，由于这些细菌酶在哺乳动物细胞中并不天然存在，细菌 Cas9 蛋白是否以及如何被哺乳动物宿主识别和调控仍知之甚少。在这里，我们鉴定出 Keap1 是一种哺乳动物内源性 E3 连接酶，它以“ETGE”-样降解基序依赖性的方式将 Cas9/dCas9/Fanzor 靶向泛素化和降解。逃避 Keap1 识别的 Cas9“ETGE”-样降解基序突变体能在细胞中显示出增强的基因编辑能力。dCas9“ETGE”-样降解基序突变体能延长蛋白半衰期，并在染色质上保持更长时间的蛋白，从而提高了 CRISPRa 和 CRISPRi 的效率。此外，Cas9 与 Keap1 的结合也通过与 Keap1 底物或结合伙伴竞争与 Keap1 的结合，从而损害了 Keap1 的功能，而工程化的 Cas9 突变体对 Keap1 生物学的干扰较小。因此，我们的研究揭示了一种哺乳动物特异性的 Cas9 调控机制，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 信号的影响最小。
