在Pichia pastoris中改进了CRISPRi系统
Shujing Qiao1,2,3, Fan Bai1,4, Peng Cai1,4
1Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023, PR China.
Synthetic and systems biotechnology
|September 11, 2023
概括
在Pichia pastoris中,一个改进的CRISPR干扰 (CRISPRi) 系统使用融合的dCas9和抑制器域进行高效的基因抑制. 通过Csy4内啡核核酶介导导向RNA处理提高了CRISPRi的效率,并使多重基因向成为可能.
科学领域:
- 分子生物学分子生物学
- 合成生物学 合成生物学
- 微生物生物技术 微生物生物技术
背景情况:
- 克里斯普尔干扰 (CRISPRi) 是各种生物体中基因抑制的强大工具.
- 在Pichia pastoris等酵母中优化CRISPRi系统对于代谢工程和功能基因组学至关重要.
研究的目的:
- 在Pichia pastoris中开发和增强CRISPRi系统,以进行强大的基因抑制.
- 研究指导RNA (gRNA) 处理对CRISPRi效率的影响.
- 通过使用工程CRISPRi系统来证明复杂基因抑制.
主要方法:
- 在Pichia pastoris中,dCas9与内源转录抑制器域的融合.
- 针对AOX1促进体和eGFP基因进行抑制.
- 使用HH/HDV ribozymes和Csy4 endoribonuclease进行gRNA处理.
- 构建gRNA数组,同时抑制多个基因.
主要成果:
- 实现了高达85%的对EGFP的镇压效率.
- 证明了原生AOX1基因的有效抑制,影响甲醇的生长.
- 表明Csy4介导的gRNA处理与原生tRNA系统相比,产生更高的抑制效率.
- 通过使用Csy4处理的gRNA阵列,同时成功抑制多个基因 (例如FAA1和FAA2).
结论:
- 在Pichia pastoris中,增强的CRISPRi系统提供了强大且可调节的基因抑制.
- Csy4内啡核酶对处理gRNA数组有效,使有效的多重基因抑制成为可能.
- 这种改进的系统为Pichia pastoris的基因操纵和合成生物学提供了宝贵的工具.
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