在Ogataea polymorpha中使用CRISPR-Cas12a/Cpf1系统的多基因精确编辑工具.
Senqin Hou1,2,3, Shibin Yang1,2, Wenqin Bai4,5
1National Center of Technology Innovation for Synthetic Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
Microbial cell factories
|January 21, 2025
概括
一个新的CRISPR-Cpf1系统显著改善了Ogataea polymorpha的基因组编辑,使有效的多基因编辑成为可能,并增强了其作为工业细胞工厂的潜力.
科学领域:
- 微生物学 微生物学
- 分子生物学分子生物学
- 合成生物学 合成生物学
背景情况:
- Ogataea polymorpha 是一种甲基转化酵母,具有产生有价值化合物的潜力.
- 有效的基因组编辑工具对于将O. polymorpha发展成细胞工厂至关重要.
- 在O. polymorpha中现有的CRISPR-Cas9系统需要改进以进行多基因编辑.
研究的目的:
- 为O. polymorpha.开发一个高效的CRISPR-Cpf1介导的基因组编辑系统.
- 为了提高同源重组 (HR) 效率,用于精确的基因组编辑.
- 为了使多重基因删除和整合能够用于代谢工程.
主要方法:
- 开发一个CRISPR-Cpf1系统用于O. polymorpha.
- 淘汰非同类末端连接 (NHEJ) 相关基因以促进HR.
- 该系统用于单基因,双基因和三基因基因编辑的应用,包括大型DNA片段操纵.
主要成果:
- 在单个 (98.1%),双重 (93.9%) 和三重 (94.0%) 基因中实现了高编辑效率.
- 通过淘汰NHEJ基因,HR效率从<30%提高到90-100%.
- 对三重基因的集成效率>90%,对20kb片段的删除>90%.
- 通过三重基因的一步整合,在O. polymorpha中成功产生了烯.
结论:
- CRISPR-Cpf1系统为O. polymorpha.中的多重基因组编辑提供了一个强大的工具.
- 该系统可有效删除和整合多个基因,加速代谢工程.
- 增强的基因组编辑能力有望在开发O. polymorpha作为工业细胞工厂方面取得进展.
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