化学介导的CRISPR/Cas9基因编辑系统在猪胚胎中使用不同的非病毒载体进行比较
Qingyi Lin1,2, Koki Takebayashi1,2, Nanaka Torigoe1,2
1Bio-Innovation Research Center, Tokushima University, Tokushima, Japan.
Animal science journal = Nihon chikusan Gakkaiho
|October 11, 2023
概括
测试了三种非病毒试剂,用于猪胚胎中集群定期间隔的短时间palindromic重复 (CRISPR) /Cas基因编辑. 虽然突变率有所改善,但胚胎细胞发育率有所下降,这表明需要进一步优化.
科学领域:
- 生殖生物学 生殖生物学
- 遗传学 是一个遗传学.
- 生物技术是生物技术.
背景情况:
- 在猪胚胎中有效的基因编辑对于农业和生物医学应用至关重要.
- 非病毒传染方法为传递CRISPR/Cas系统的病毒载体提供了潜在的替代方案.
研究的目的:
- 为了比较三种非病毒传染试剂 (TransIT-X2,CRISPRMAX,ProteoCarry) 在将CRISPR/Cas核糖蛋白复合物输送到猪胚胎和胚胎中的效率.
- 优化Cas9:gRNA比率,试剂剂量和转染时间,以最大限度地提高基因编辑效率,同时最大限度地减少对胚胎发育的影响.
主要方法:
- 使用三种非病毒试剂,将猪的阴茎转化为CRISPR相关蛋白9 (Cas9) 和指导RNA (gRNA) 向GGTA1.
- 实验涉及优化Cas9:gRNA分子比率 (1:2, 2:2, 4:2),试剂剂量 (单/双),以及转染时间 (IVF后10小时对29小时).
- 测量结果包括囊胚细胞形成率,突变率和突变效率.
主要成果:
- 一个4:2 Cas9:gRNA分子比和双试剂剂量通常会增加所有试剂的突变率.
- 然而,与对照组相比,更高的突变率与胚胎细胞形成率下降的趋势相关.
- 最佳的转染时间因试剂而异,在所有测试条件下,突变芽细胞的比例低于34%.
结论:
- 这三种非病毒转染试剂有助于在猪的胚胎和胚胎中进行基因编辑.
- 目前基于化学的转染技术需要在基因编辑效率和早期胚胎发育方面显著改进,以便在实践中应用.
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