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基于小型化的Cas蛋白的CRISPR/Gal4BD-Cas捐赠者适应系统,用于改进基因编辑

Sen Yang¹, Bao-Xia Ma¹, Hong-Run Qian¹

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

Yi chuan = Hereditas

|September 14, 2024

概括

这项研究引入了基于CRISPR的小型化捐赠者适应系统 (DAS),用于增强基因编辑. 这些系统提高了哺乳动物细胞的同质导向修复 (HDR) 效率,推进了精密医学和育种应用.

科学领域:

分子生物学分子生物学
基因编辑技术的技术
生物技术是生物技术.

背景情况:

同源导向修复 (HDR) 可以实现精确的基因编辑,但由于在双链断裂 (DSB) 时捐赠DNA的招募不佳,其效率较低.
之前的CRISPR/Cas9-Gal4BD捐赠者适应系统 (DAS) 面临着SpCas9蛋白大小的局限性,阻碍表达,包装和体内输送.
微型化Cas蛋白为改善DAS的实用性和有效性提供了潜在的解决方案.

研究的目的:

开发和验证使用SlugCas9和AsCas12a.的新型小型化CRISPR/Gal4BD供体适应系统 (DAS).
评估Gal4BD融合对小型化Cas蛋白的活性的影响.
优化捐赠者DNA模板配置,以提高HDR效率,使用新的DAS.

主要方法:

通过将小型化的 Cas 蛋白与 Gal4BD 适配器融合,开发出了 CRISPR/Gal4BD-SlugCas9 和 CRISPR/Gal4BD-AsCas12a DAS.
使用SSA (桑格序列分析) 报告测试来评估Cas-Gal4BD融合的向活动.
雇佣的HDR效率报告员测试和优化dsDNA模板设计 (BS-dsDNA和dsDNA-BS) 进行功能验证.

主要成果:

4BD与SlugCas9和AsCas12a的N终端的融合对其准活动的影响最小.
优化的捐赠者模式提高了HDR效率;BS-dsDNA对CRISPR/Gal4BD-AsCas12a更优越,而dsDNA-BS则被推用于CRISPR/Gal4BD-SlugCas9.
在HEK293T细胞中的EMX1,NUDT5和AAVS1位点,CRISPR/Gal4BD-SlugCas9 DAS实现了显著的基因编辑效率 (24-37%).

结论:

新的CRISPR/Gal4BD-SlugCas9和CRISPR/Gal4BD-AsCas12a DAS有效地提高了HDR介导的基因编辑效率.
这项研究为优化未来的捐赠者适应系统提供了宝贵的见解.
这些进步扩大了基因编辑的工具包,特别是动物分子设计育种和精密医学.

关键词:

适配器适配器适配器适配器供体适应供体适应基因编辑基因编辑同质性导向的维修是指导维修.微型化的Cas蛋白质.

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