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Updated: May 17, 2025

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通过SSODN通过HDR进行高效的GBA1编辑,通过在CRISPR/Cas9裂变上超越伪基因介导的基因转换

Joseph S Lagas¹, Monica F Sentmanat¹, Xiaoxia Cui¹

¹Department of Genetics, Genome Engineering and Stem Cell Center at the McDonnel Genome Institute (GESC@MGI), School of Medicine, Washington University in St. Louis, St. Louis, MO, United States.

Frontiers in genome editing

|May 15, 2025

概括

在CRISPR/Cas9编辑中的基因转换可以通过单链寡氧核酸 (ssODN) 捐赠者来克服. 这种方法可以在基因伪基因对中进行精确的基因编辑,这对于使用诱导多能干细胞 (iPSC) 进行疾病建模至关重要.

科学领域:

分子生物学分子生物学
基因编辑技术的技术
干细胞研究干细胞研究

背景情况:

在使用诱导多能干细胞 (iPSC) 创建疾病模型时,CRISPR/Cas9基因编辑至关重要.
非常相似的基因伪基因对之间的基因转换对精确的基因编辑构成重大挑战.
β-葡萄糖核糖酶基因 (GBA1) 的突变与神经退行性疾病 (如帕金森病和高氏病) 有关.

研究的目的:

开发一种方法来精确编辑 iPSC 中的 GBA1 的基因,克服其伪基因 (GBAP1) 所带来的挑战.
为了提高同质导向修复 (HDR) 在CRISPR/Cas9介导的双链断裂修复过程中对基因转换的效率.
建立一个可靠的策略来产生双基淘汰 (KO) iPSC线路用于疾病研究.

主要方法:

使用CRISPR/Cas9诱导iPSC中的GBA1基因发生双链断裂.
单链寡度氧核酸 (ssODN) 捐赠者被提供为同质导向修复 (HDR) 模板.
评估了HDR在与基因转换和非基同源重组 (NAHR) 竞争中的效率.

主要成果:

从GBA1伪基因 (GBAP1) 的基因转换发生在大约70%的编辑事件中,保持了阅读框架.
引入ssODN捐赠体显著增加了通过HDR带有外框架indels的等位基因的百分比.
这种以HDR为重点的方法成功地使双基GBA1KO iPSC克隆的分离成为可能.

结论:

外源的ssODN模板可以有效地将DNA修复引导到HDR路径上,从而超越了基因转换.
这种可通用的方法允许在基因伪基因对中进行特定的基因编辑,而不会影响伪基因.
该策略对于生成准确的iPSC模型来研究与GBA1相关的疾病至关重要.

关键词:

基因转换 (GC) 是一种基因转换.基因编辑 (CRISPR/Cas9) 是一种技术.敲打出去,敲打进来.非基同源重组 (NAHR) 的方法伪基因伪基因伪基因伪基因干细胞工程干细胞工程

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