工程 Cas9 变种绕过 Keap1 介导的人类细胞的降解,并提高表观基因组编辑效率
Jianfeng Chen1,2, Siyuan Su1,2, Adrian Pickar-Oliver3,4
1Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Nucleic acids research
|September 4, 2024
概括
哺乳动物细胞利用Keap1,一种新发现的E3结合酶,准细菌Cas9蛋白进行降解. 改造Cas9变种以逃避Keap1增强基因编辑和CRISPR应用,同时尽量减少对宿主细胞信号的干扰.
科学领域:
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
- 生物化学 生物化学
背景情况:
- Cas9是一种强大的基因组编辑工具,广泛用于研究和治疗.
- 细菌Cas9蛋白质在哺乳动物细胞中自然存在,导致对宿主调节的理解较差.
- 了解Cas9宿主相互作用对于优化其治疗应用至关重要.
研究的目的:
- 确定调节Cas9及其变体的哺乳动物蛋白质.
- 研究Cas9在哺乳动物细胞中的泛化和降解机制.
- 设计具有提高效率和减少对宿主通路的目标外影响的Cas9变体.
主要方法:
- 同免疫沉测试以确定Cas9相互作用蛋白质.
- 乌比基化试验用于评估Cas9的修饰.
- 在哺乳动物细胞中进行CRISPR基因编辑,激活 (CRISPRa) 和干扰 (CRISPRi) 试验.
- 对Cas9蛋白半衰期和染色质保留的分析.
主要成果:
- 哺乳动物E3结合酶Keap1针对Cas9,dCas9和Fanzor通过类似ETGE的降解来降解.
- 具有"ETGE"类降解基因突变的Cas9变体显示了基因编辑效率的提高.
- 经过修改的降解子的dCas9变体表现出更长的半衰期和更好的染色质保留,促进CRISPRa和CRISPRi.
- Cas9与Keap1结合可以破坏Keap1的正常功能;工程突变者将这种破坏降到最低.
结论:
- 凯普1调节细菌Cas9蛋白的哺乳动物特异性调节.
- 设计Cas9变种以逃避Keap1的识别,提高它们的基因编辑和调控能力.
- 开发的Cas9设计在CRISPR应用中提供了更高的效率,对内源Keap1信号通路的影响减少.
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