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一个高效的CRISPR/Cas9基因组编辑系统,基于Trichoderma reesei中的多个sgRNA处理平台,用于菌株改进和酶生产

Jiaxin Zhang¹, Kehang Li¹, Yu Sun¹

¹State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, Qingdao, 266237, People's Republic of China.

Biotechnology for biofuels and bioproducts

|February 11, 2024

概括

使用tRNA-sgRNA数组的新CRISPR-Cas9系统可以在Trichoderma reesei中进行多重基因组编辑. 这一突破增强了酶和重组蛋白质的产生,加速了真菌菌株的改善.

科学领域:

分子生物学分子生物学
生物技术是生物技术.
菌类遗传学菌类遗传学

背景情况:

克里斯普尔/Cas9是Trichoderma reesei基因工程的一个关键工具.
在T. reesei中,多重基因编辑受到单向导RNA (sgRNA) 处理的限制.
这种限制阻碍了酶和蛋白质生产的菌株开发.

研究的目的:

开发一个CRISPR/Cas9系统用于T. reesei多重基因组编辑.
克服sgRNA处理中的局限性,用于增强菌株工程.
为了提高降解纤维素酶和重组蛋白质的生产.

主要方法:

建立了一个使用arrayed tRNA-sgRNA架构的CRISPR/Cas9系统.
使用5SrRNA促进器从单个转录生成多个sgRNA.
采用内源tRNA处理系统进行sgRNA成熟.

主要成果:

使用两个sgRNAs成功删除了cre1基因,证明了高效的处理.
通过ace1位点修饰,通过ace1位点修饰实现了增强的细胞酶和西兰酶生产 (高达18倍和41倍).
通过删除cbh1和cbh2基因,设计了一种高水平的葡萄糖氧化酶生产 (43.77 U/mL) 的菌株.

结论:

基于tRNA-gRNA阵列的CRISPR-Cas9系统对T. reesei基因组编辑非常有效.
这种系统加速了T. reesei的工程,以生产有价值的酶和蛋白质.
扩大了用于真菌合成生物学和基因组工程的CRISPR工具箱.

关键词:

这就是CRISPR/Cas9的作用.葡萄糖氧化酶是什么?红纤维素降解酶的降解酶多基因编辑多基因编辑处理多个sgRNA处理.三甲虫reesei 在线阅读

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