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迷你PE,一个主要编辑器与紧的Cas9和截断的逆转录酶

Ting Lan^1,2, Huangyao Chen^1,3,2, Chengcheng Tang¹

¹Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Wuyi University, Jiangmen 529020, China.

Molecular therapy. Nucleic acids

|September 8, 2023

概括

研究人员通过优化Moloney小鼠白血病病毒 (MMLV) 逆转录酶 (RT) 来开发了一个迷你主要编辑器 (PE). 这种较小的PE工具可以在细胞和小鼠视网膜中精确编辑基因,为潜在的遗传疾病治疗铺平了道路.

科学领域:

分子生物学分子生物学
基因编辑技术的技术
生物技术是生物技术.

背景情况:

主编辑 (PE) 是一种强大的基因组编辑技术,可以在不需要DNA捐赠者或诱导双链断裂的情况下进行精确的DNA修改.
目前主要编辑器系统的大小对高效的体内传输和应用构成了重大挑战.
开发更小,更易于管理的原始编辑器变体对于推进体内基因编辑疗法至关重要.

研究的目的:

设计一个小型化主编辑器 (mini-PE) 系统,为体内应用程序提供增强的交付能力.
识别和优化主要编辑器的关键组件,特别是反转录酶 (RT) 域.
在细胞和动物模型中评估开发的迷你PE的效率和精度.

主要方法:

截断的莫洛尼小鼠白血病病毒 (MMLV) 反转录酶 (RT) 变体的查,包括具有F155Y突变的变体,以确定较小,高效的组件.
优化pegRNAs和纳入nicking sgRNAs,以增强迷你PE系统的编辑活动.
在小鼠模型中使用腺相关病毒 (AAV) 进行体内基因编辑的迷你PE系统的交付.

主要成果:

确定了最小的功能性截断的MMLV RT变种,保留了高基因编辑效率.
优化的迷你PE系统在人类和小鼠细胞的目标部位实现了高达10%的精确编辑.
通过使用AAV传递在视网膜中成功编辑小鼠HSF1基因,尽管效率低于1%.

结论:

通过优化MMLV RT,成功开发了小型化主要编辑器 (mini-PE),使得在体外有效的基因编辑成为可能.
迷你PE系统对体内基因编辑应用有前途,如老鼠视网膜编辑所示.
需要进一步提高编辑效率,才能充分利用迷你PE对人类遗传疾病的治疗潜力.

关键词:

在 AAV AAV AAV 中.这就是CjCas9的原因.MT:RNA/DNA编辑编辑这是一个迷你的迷你.总编辑总编辑总编辑截断的MMLV可以使用.

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