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使用CRISPR/Cas9-Gal4系统提高羊初级细胞中的基因突击效率

Yan Li¹, Sujun Wu², Yunpeng Wu¹

¹Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100071, China.

Current issues in molecular biology

|November 26, 2025

概括

研究人员通过增加DNA修复模板度,改善了羊的CRISPR基因编辑. 这提高了羊胎儿纤维细胞中大DNA片段的敲进效率,有助于农业和生物医学应用.

科学领域:

* 分子生物学 * 分子生物学
* 遗传学在遗传学方面
* 生物技术 * 生物技术

背景情况:

*在绵羊中通过CRISPR进行基因组编辑,面临的挑战是大DNA片段集成效率低.
* 这种局限性阻碍了在农业,生物学和生物医学中用于羊的精确基因编辑应用.
*目前的方法通常依赖于药物选择或光丰富,这并不总是理想的.

研究的目的:

* 提高使用CRISPR技术在绵羊中针对大型外源DNA片段的有针对性整合的效率.
* 开发一种更易于使用和更有效的方法,用于在羊群中使用CRISPR介导的基因敲定.
* 为了克服羊胎儿纤维细胞中低敲进效率的局限性.

主要方法:

*采用一种策略,在DNA双链断裂 (DSB) 部位增加同源修复模板的局部度.
*通过柔性链接器将Gal4蛋白 (Gal4-BD) 的DNA结合域 (BD) 与SpCas9蛋白的N端结合在一起.
* 在捐赠模板的3'端包含一个UAS序列,用于Gal4-BD.的特定结合.

主要成果:

* 大量的外源DNA片段 (2997 bp) 的敲进效率得到了显著改善.
*羊胎儿纤维细胞 (SFFs) 的内置效率从5.30% (8/151) 增加到16.67% (32/192).
* 展示了一种易于使用的方法,用于在绵羊中有效的CRISPR介导基因内置.

结论:

* 开发的方法有效地提高了大型DNA片段在绵羊中敲击的效率.
* 这一策略为推进在绵羊中通过CRISPR介导的基因组编辑提供了一个有价值的工具.
*这些发现支持精确基因编辑在羊群中的更广泛应用,用于各种科学和农业目的.

关键词:

在CRISPR/Cas9中使用.盖尔4 盖尔4 盖尔4 是一个同质性导向的维修是指导维修.羊的胎儿纤维细胞.

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