关于在人类多能干细胞中进行原始编辑的设计,实施和评估的协议
Youjun Wu1, Mega Sidharta1, Aaron Zhong1
1The SKI Stem Cell Research Facility, The Center for Stem Cell Biology and Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, New York, NY 10065, USA.
STAR protocols
|September 22, 2023
概括
这项研究介绍了人类干细胞中主要编辑的协议,使得精确的基因修改,如单核酸变化没有DNA断裂. 该方法通过纠正特定的GBA突变来验证.
科学领域:
- 遗传学 是一个遗传学.
- 分子生物学分子生物学
- 干细胞研究 干细胞研究
背景情况:
- 主编辑提供精确的基因组编辑,没有双链DNA断裂.
- 现有的协议需要针对特定的细胞类型进行优化,例如人类多能干细胞.
研究的目的:
- 建立和验证人类多能干细胞中主要编辑的综合性协议.
- 为了证明原始编辑在引入和纠正特定突变方面的效率.
主要方法:
- 主编辑指导RNA (pegRNA) 和nicking sgRNA的设计和克隆.
- 在人类多能干细胞中,主要编辑组件的电穿孔.
- 使用Miseq测序进行效率评估.
- 适用于GBA (N370S) 突变诱导和纠正.
主要成果:
- 在人类多能干细胞中成功实施了主要编辑协议.
- 证明了引入和纠正GBA (N370S) 突变的能力.
- Miseq分析提供了编辑效率的定量评估.
结论:
- 开发的协议是有效的精确基因组编辑在人类多能干细胞.
- 总编辑为基因工程和干细胞治疗应用提供了一种多功能工具.
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