在猪纤维细胞中的多个基因的CRISPR核糖蛋白介导精确编辑
Xiaochen Guo1,2, Chang Liu1,2, Yunjing Zhao1
1Key Laboratory of Animal Cellular and Genetic Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin 150030, China.
Animals : an open access journal from MDPI
|February 24, 2024
概括
这项研究增强了使用M3814和酸修饰的ssODN在猪细胞中的CRISPR RNP基因编辑. 优化的方法实现了高效,多基因编辑,以加速遗传繁殖.
科学领域:
- 分子生物学分子生物学
- 遗传学 是一个遗传学.
- 生物技术是生物技术.
背景情况:
- 在猪细胞中开发高效的多基因编辑策略对于遗传育种至关重要.
- 目前的方法对于猪的同时精确的多基因编辑缺乏有效性.
研究的目的:
- 为了提高猪细胞中CRISPR RNP介导的精确基因编辑效率.
- 建立一种在猪细胞中同时进行多基因编辑的方法.
主要方法:
- 利用CRISPR RNP (Cas9蛋白,sgRNA,ssODN) 在猪胎儿纤维细胞 (PFFs) 中进行同质导向修复 (HDR).
- 通过电穿孔引入组件,并使用M3814和酸改性ssODN (PS-ssODN) 增强HDR.
- 通过DNA测序分析编辑效率,并比较各种CRISPR RNP系统组合.
主要成果:
- 使用2μM M3814的CRISPR RNP使HDR效率提高了3.62倍,而没有PFF毒性.
- 克里斯普尔RNP,M3814和PS-ssODNs的组合实现了42.81%的HDR效率,增加了6.38倍.
- 在猪细胞中成功地同时编辑了两个和三个位点 (INS,RLN3外因子1,RLN3外因子2).
结论:
- 优化的CRISPR RNP系统与M3814和PS-ssODNs显著提高了猪细胞中精确基因编辑的有效性.
- 这种方法可以同时精确编辑多达三种基因,推进遗传育种应用.
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