在诱导多能干干细胞中进行精确基因组编辑的CRISPR/Cas9基协议
Avinash Singh1,2, Swathy Babu1,2, Marcus Phan1,3
1Department of Neurology, University of Minnesota, Twin Cities, Minneapolis, MN, USA.
Bio-protocol
|December 30, 2024
概括
通过抑制p53和使用亲存活分子,在人类诱导多能干细胞 (iPSCs) 中增强CRISPR基因组编辑. 这种方法可以提高细胞存活率,并达到90%以上的同源重组效率.
科学领域:
- 分子生物学分子生物学
- 干细胞生物学 干细胞生物学
- 基因工程是一种基因工程.
背景情况:
- 克里斯普尔/卡斯9技术提供了强大的基因组编辑能力.
- 使用CRISPR编辑人类诱导的多能干细胞 (iPSCs) 面临着细胞生存和效率方面的挑战.
- 在CRISPR编辑过程中激活P53可以诱导细胞亡,阻碍成功.
研究的目的:
- 使用CRISPR开发一种协议,以提高人类iPSC细胞的细胞存活率和编辑效率.
- 为了减轻由CRISPR编辑引发的亡反应.
- 为了简化同源细胞系的产生.
主要方法:
- 在CRISPR编辑过程中将p53抑制与支持生存的小分子结合起来.
- 使用CRISPR/Cas9技术对人类iPSC细胞进行向基因组修改.
- 评估同源重组率和编辑后的细胞存活率.
主要成果:
- 一个结合p53抑制和亲存活分子的新方案显著提高了细胞存活率.
- 在人类iPSC中达到超过90%的同源重组率.
- 将iPSC基因组编辑的总体时间缩短到8周.
结论:
- 抑制p53和促生存分子的组合是改善iPSC中的CRISPR编辑结果的有效策略.
- 这种优化的协议加速了转基因iPSCs的生成.
- 这些发现有助于有效地创建异构细胞系,用于研究和治疗应用.
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