利用内源性I-F型CRISPR/Cas系统,有效地进行基因工程和基因抑制,在Pseudomonas chlororaphis LX24中
Yan-Fang Nie1, Sheng-Jie Yue1, Peng Huang1
1State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
ACS synthetic biology
|September 18, 2025
概括
研究人员为Pseudomonas chlororaphis LX24开发了一个新的遗传工具包,提高了其代谢工程能力. 该系统可实现精确的基因组编辑和转录抑制,释放了农业和工业应用的潜力.
科学领域:
- 微生物学和分子生物学
- 合成生物学 合成生物学
- 农业生物技术 农业生物技术
背景情况:
- Pseudomonas chlororaphis 是一种促进植物生长的草原细菌,具有重要的农业和工业潜力.
- 在此之前,有限的遗传工具阻碍了P. chlororaphis.的代谢工程工作.
- 内生CRISPR/Cas系统为开发非模型生物体中的新型遗传工具提供了一个有希望的途径.
研究的目的:
- 描述和利用P. chlororaphis LX24中的内源型I-FCRISPR/Cas系统进行基因组编辑.
- 通过克服限制-修改系统来提高等离子体转换效率.
- 开发一套多功能基因工具包,包括CRISPR干扰 (CRISPRi),用于精确的基因操纵.
主要方法:
- 一个内源型I-FCRISPR/Cas系统的表征和可编程基因组编辑工具包的开发.
- 识别和删除限制修改系统以提高等离子体转换效率.
- 实施 λ-Red 重组和基于 sacB 的反选择,以实现高效的基因淘汰和等离子体治愈.
- 通过敲除Cas3核酶,建立一个CRISPR干扰 (CRISPRi) 系统.
主要成果:
- 为P. chlororaphis LX24建立了一个高效的基因组编辑工具包,实现了22-87%的编辑效率.
- 使用λ-红色重组,纳集群的基因淘汰效率增加了9倍以上.
- 成功开发了CRISPRi系统,使可调节的转录抑制氨酸生产 (减少21-89%).
- 实现了单步基因插入和替换,成功率为100%.
结论:
- 已经开发出了P. chlororaphis LX24的方便和精确的遗传工具,大大推进了其代谢工程.
- 开发的方法为在其他非模型 prokaryotes 中重新利用内源 CRISPR 系统提供了有价值的参考.
- 该工具包通过有针对性的基因改造增强了P. chlororaphis在农业和工业应用中的潜力.
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