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基于CRISPR-Cas9的一步多重基因组编辑,通过优化Pichia pastoris中的指导RNA处理策略

Kaidi Chen^1,2, Gulikezi Maimaitirexiati¹, Qiannan Zhang¹

¹Center for Synthetic Biochemistry, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), Shenzhen, 518055, China.

Synthetic and systems biotechnology

|February 25, 2025

概括

研究人员开发了一个CRISPR/Cas9系统,用于在Pichia pastoris中高效的多基因编辑. 这种工具可以快速实现双基因淘汰和多基因集成,促进其在代谢工程中用于化合物生产的应用.

科学领域:

生物技术是生物技术.
合成生物学合成生物学
微生物工程微生物工程

背景情况:

皮奇亚牧师是一种甲基转化酵母,具有从甲醇中产生重组蛋白和增值化合物的显著潜力.
目前基因组工程工具的局限性阻碍了P. pastoris.中复杂的代谢工程应用.
需要有效的工具来增强P. pastoris作为C1生物利用的细胞工厂.

研究的目的:

使用CRISPR/Cas9.9开发一个快速高效的多基因编辑系统,用于Pichia pastoris.
优化指导RNA处理策略,以提高基因编辑效率.
通过代谢工程来证明系统在单步化合物生产中的实用性.

主要方法:

优化单导引RNA (sgRNA) 处理盒,包括tRNA-sgRNA-tRNA (tgt),HgH (HH-sgRNA-HDV) 和tRNA-sgRNA-HDV (tgH) 结构.
开发一种双HgH (dHgH) 结构,用于一阶段的双基因破坏和多基因集成.
开发的系统的应用用于脂肪酸和5-基酸的一步生产.

主要成果:

HgH结构表现出最高的单基因淘汰效率,为95.8%.
该dHgH结构使得高效的一步双基因淘汰 (60-100%) 和多基因集成.
取得了成功的一步生产脂肪酸 (23 mg/L/μg蛋白/OD) 和5-基酸 (13.3 mg/L).

结论:

基于CRISPR/Cas9的多基因编辑系统显著提高了Pichia pastoris的基因组工程能力.
这个系统促进了复杂的代谢工程策略,增强了P. pastoris作为细胞工厂.
开发的工具对于推进C1生物利用和多重复合物生物合成有价值.

关键词:

这就是CRISPR/Cas9的作用.基因删除和整合.基因组编辑基因组编辑这就是Pichia pastoris.在gRNA处理过程中.

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