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使用高效的PEG10修饰构建一个TAT-Cas9-EGFP位点特异整合的真核细胞系

Shiyu Qi¹, Yibo Wang¹, Zhimei Liu¹

¹Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China.

International journal of molecular sciences

|February 13, 2025

概括

这项研究通过修改5'末端PEG10来增强CRISPR/Cas9基因编辑,显著提高同质导向修复敲门效率,用于创建疾病模型并改善蛋白质生产.

科学领域:

分子生物学分子生物学
基因编辑技术的技术

背景情况:

克里斯普尔/Cas9对于真核生物基因组修改至关重要,同质导向修复 (HDR) 敲门 (KI) 对于疾病建模和基因疗法至关重要.
低HDR介导的KI效率是基因工程应用中的一个重大局限.
HEK293细胞,特别是293T细胞系,对于重组蛋白质的产生非常有价值,特别是当在AAVS1位点进行特定位点的整合以获得稳定的表达时.

研究的目的:

研究5'端PEG10修饰对特定位点基因整合效率的影响.
提高293T细胞AAVS1位点的同质导向修复 (HDR) 中介敲进 (KI) 效率.
为了建立一个高表达,特定站点的集成293T细胞系用于TAT-Cas9-EGFP生产.

主要方法:

使用了具有5'末端PEG10修饰的CRISPR/Cas9系统.
在293T细胞系的AAVS1位点进行了特定位点的基因整合.
对1.8KB的目标碎片进行评估的敲门效率.

主要成果:

5'端PEG10修改增加了1.9倍的敲进效率,对于1.8kb的目标片段.
随着修改,内置效率从26%提高到49%.
成功建立了一个高表达性,特定位点的集成293T细胞系,用于TAT-Cas9-EGFP.

结论:

5'端的PEG10修改显著提高了HDR介导的KI效率.
这种优化的系统提供了一种可靠的方法,用于创建稳定,高表达的细胞系用于蛋白质生产.
开发的293T细胞系作为重组蛋白制造的宝贵资源.

关键词:

这就是CRISPR/Cas9的作用.这就是HDR HDR.效率 KI 的效率 KI 的效率.在PEG10的基础上,PEG10 这就是TAT TAT.

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