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优化CRISPR/Cas9核糖核蛋白的传递,以便在牛胚胎中有效编辑基因组

Xiaofeng Du¹, Alexander Quinn¹, Moira Menzies¹

¹Commonwealth Scientific and Industrial Research Organisation, Agriculture & Food, Brisbane, QLD 4072, Australia.

|August 8, 2025

概括

我们比较了CRISPR-Cas9传递方法用于基因编辑牛胚胎. 电穿孔实现了最高的编辑效率 (65.2%),但影响了胚胎发育,为牲畜基因组编辑工作流提供了洞察力.

科学领域:

动物生物技术动物生物技术
分子生物学分子生物学
发展生物学发展生物学

背景情况:

在牲畜中有效的基因编辑对于农业的进步至关重要.
克里斯普尔-Cas9技术提供了精确的基因组修改能力.
为成功的基因编辑,优化将CRISPR-Cas9组件输送到牛肉囊中的输送方法至关重要.

研究的目的:

评估和比较三种不同的转染方法,用于将CRISPR Cas9-sgRNA核糖蛋白输送到牛肉细胞内.
评估基因编辑的效率及其对每个方法胚胎发育的影响.
确定最佳策略,以简化牛胚胎中的基因编辑工作流程.

主要方法:

使用Lipofectamine CRISPRMAX和使用Neon和NEPA21系统的电穿孔进行了测试.
博斯金牛的益生菌受体 (PRLR) 基因是编辑的目标.
基因编辑结果通过PCR基因定型和桑格测序在胚胎囊阶段 (8日后受精) 分析.

主要成果:

在CRISPRMAX的脂化中,高达30%的PRLR编辑的芽细胞产生,对裂变 (93%) 和芽细胞率 (39%) 的影响最小.
电穿孔参数 (电压,脉冲持续时间,数量) 影响了编辑效率,但降低了胚胎活力.
电穿孔实现了最高的编辑效率 (65.2%编辑了胚胎细胞,21%是同卵性),但导致了较低的裂变率 (50%) 和胚胎细胞率 (10%).
NEPA21电穿孔,特别是用增强剂,达到了47.6%的编辑胚胎,而将NEPA21与CRISPRMAX脂肪移植结合在一起,导致50%的编辑胚胎 (23%的同卵性) 与单独NEPA21相比改善了胚胎发育.

结论:

不同的CRISPR-Cas9传递方法显示出不同的效率和对牛胚胎发育的影响.
电穿孔提供了高的编辑效率,但需要优化,以平衡与胚胎的生存能力.
这些发现为在牲畜中开发高效的基因编辑工作流提供了宝贵的见解,有可能加速基因组编辑技术的采用.

关键词:

牛类牛类牛类牛类牛类牛这就是CRISPR/Cas9的作用.效率效率是指效率是指效率.胚胎是如何形成的基因编辑基因编辑转化转化转化转化

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