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工程 TALE-链接除氨酶以促进线粒体DNA中的精密腺基编辑

Sung-Ik Cho¹, Kayeong Lim², Seongho Hong³

¹Center for Genome Engineering, Institute for Basic Science, Daejeon 34126, Republic of Korea; Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea; Department of Pharmacology, Yonsei University College of Medicine, Seoul 03722, Republic of Korea.

|January 5, 2024

概括

工程基因编辑器显著减少不必要的RNA和DNA编辑,提高了线粒体基因治疗和疾病建模的安全性. 这一突破在细胞和动物模型中最大限度地减少了非目标突变和细胞毒性.

科学领域:

分子生物学
遗传学
生物技术

背景情况:

线粒体DNA (mtDNA) 编辑对遗传疾病具有前景.
目前的基编辑器,如DddA衍生的细胞基编辑器 (DdCBEs) 和转录激活器类效应体 (TALE) 相关的除氨酶 (TALEDs) 能够进行针对性的mtDNA修改.
TALEDs,特别是A-to-G编辑器,在人类细胞中显示出显著的非目标RNA编辑.

研究的目的:

设计具有减少非目标RNA编辑的TALED变体.
提高线粒体应用的基数编辑的安全性和有效性.
开发一种更精确的工具来模拟线粒体疾病,并探索治疗策略.

主要方法:

设计TadA8e的基质结合部位,这是TALED的除氨酶组成部分.
开发具有微调除氨酶活性的TALED变体.
评估转录组范围内的非目标编辑,mtDNA突变和旁观者编辑.
在小鼠胚胎中评估细胞毒性和发育影响.
产生具有致病性mtDNA突变的小鼠模型.

主要成果:

工程 TALED 变种减少了 99% 以上的转录组范围的非目标编辑.
在目标部位的mtDNA突变和旁观者编辑被最小化.
在小鼠胚胎中,TALED变体的细胞毒性降低,并没有导致发育停止.
具有致病性mtDNA突变 (利氏综合征) 的小鼠成功生成并显示心率降低.

结论:

工程 TALED 变种为线粒体基编辑提供了更安全,更精确的方法.
这些改进的工具适用于模拟线粒体遗传疾病和开发治疗干预措施.
开发的TALED变种克服了以前的基准编辑器的局限性,使得体内应用具有较低的风险.

关键词:

基因编辑器李氏综合征脱离目标的RNA 与TALE相关的腺素脱氨酶时间表遗传性疾病在体内基因组编辑线粒体线粒体基因组编辑它们的 mtDNA

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