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在Ganoderma lucidum中使用Cas9-gRNA核糖核蛋白介导基因编辑向插入异质DNA

Hyerang Eom¹, Yeon-Jae Choi¹, Rutuja Nandre¹

¹Department of BioMedical Bigdata (BK21) and Research Institute of Life Sciences, Gyeongsang National University, Jinju, Republic of Korea.

|January 29, 2025

概括

这项研究表明,同质定向修复 (HDR) 对于在中精确插入基因是有效的. 研究人员在Ganoderma lucidum中实现了15.6%的HDR效率,从而实现了针对性基因编辑以增强真菌功能.

科学领域:

菌类学菌类学是指菌类学.
分子生物学分子生物学
生物技术是生物技术.

背景情况:

CRISPR/Cas9基因编辑是功能化的强大工具.
非同源端结合 (NHEJ) 是常见的基因破坏,但通过同源导向修复 (HDR) 精确的DNA插入在真菌中较少被探索.

研究的目的:

评估在中在CRISPR/Cas9诱导的双链断裂 (DSB) 中插入具有或没有同源臂的供体DNA的疗效.
为了确定HDR在Ganoderma lucidum中精确插入基因的效率.

主要方法:

捐赠者DNA (8-1008 bp) 与Cas9-gRNA核糖核蛋白 (RNP) 复合体共同转化,这些复合体向Ganoderma lucidum中的pyrG基因.
捐赠者DNA与没有300bp同源臂 (HR_donor DNA) 进行了使用.
对变体进行了限制分析和测序,以确认供体DNA的整合,并评估HDR效率.

主要成果:

转换产生了 184 个没有同源臂的变换器和 781 个具有 HR_donor DNA 的变换器.
122个变换剂通过HR显示出完整的供体DNA序列,达到15.6%的HDR效率 (122/781).
从没有同类臂的变压器中观察到只有8例NHEJ介导的集成.

结论:

同源性定向修复 (HDR) 是一种可行的策略,用于在中精确的基因组编辑.
这种方法可以进行有针对性的修改,以引入新的真菌功能.
这项研究为在物种中进行先进基因工程奠定了基础.

关键词:

草皮虫 (Ganoderma) 是一种草皮虫.同质导向修复 (HDR) 是指同质导向的修复.基因编辑基因编辑是一种真菌.非同类末端连接 (NHEJ)

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