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Updated: Jun 23, 2025

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Published on: May 25, 2018

对不同的CRISPR/Cas9基因编辑供体适应系统进行比较和优化

Bao-Xia Ma¹, Sen Yang¹, Ming Lyu¹

¹College of Animal Science and Technology, Northwest A&F University, Yangling 71200, China.

Yi chuan = Hereditas

|June 17, 2024

概括

通过将适配蛋白与SpCas9融合,提高了CRISPR/Cas9基因编辑效率,从而增强了同质导向修复 (HDR) 以实现成功的基因敲门. 特定的适应器系统显著提高了目标基因的内置率.

科学领域:

分子生物学分子生物学
基因编辑技术的技术
基因组学就是基因组学.

背景情况:

同源导向修复 (HDR) 对于哺乳动物细胞中的基因敲定至关重要.
低HDR效率,通常由DNA模板和目标站点共定位限制,阻碍了基因编辑.
与适应蛋白融合的CRISPR/Cas9系统有可能增强HDR.

研究的目的:

为了比较和优化各种CRISPR/Cas9捐赠者适配器基因编辑系统.
通过CRISPR/Cas9.9调解基因敲进的效率提高.
研究适配蛋白融合对SpCas9活动和HDR效率的影响.

主要方法:

合适应蛋白 (yGal4BD,hGal4BD,hLacI,hTHAP11,N57) 与SpCas9.9结合在一起
利用优化的捐赠者DNA模板设计用于eGFP基因敲进.
通过PCR和桑格测序评估了敲门精度;通过HEK293T细胞的流细胞计评估了效率.

主要成果:

适应蛋白的融合并没有显著影响SpCas9的活性.
与yGal4BD,hGal4BD,hLacI和hTHAP11融合的SpCas9显著提高了GAPDH部位的敲进效率.
与yGal4BD和hGal4BD融合的SpCas9显著提高了ACTB站点的敲进效率;增加了BS增强的SpCas9-hTHAP11介导的敲进.

结论:

克里斯普尔/卡斯9捐赠者适配系统,特别是那些具有yGal4BD和hGal4BD的系统,提高了基因敲进效率.
优化的捐赠者DNA模板设计和适配蛋白融合是有效基因编辑的关键.
这项研究为推进CRISPR/Cas9介导基因编辑应用提供了宝贵的见解.

关键词:

这就是CRISPR/Cas9的作用.供体适应供体适应基因编辑基因编辑基因敲进式的基因敲进式同质性导向的维修是指导维修.

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