CRISPR/Cas9-mediated genome editing directed by a 5S rRNA-tRNA(Gly) hybrid promoter in the thermophilic filamentous fungus Humicola insolens

文献类型： 外文期刊

第一作者： Fan, Chao

作者： Fan, Chao;Zhang, Wei;Ji, Wangli;Zhang, Yuhong;Liu, Bo;Xu, Xinxin;Su, Xiaoyun;Luo, Huiying;Yao, Bin;Huang, Huoqing

作者机构：

关键词： Humicola insolens; CRISPR; Cas9; 5S rRNA; tRNA(Gly); Cellulase

期刊名称：BIOTECHNOLOGY FOR BIOFUELS （ 影响因子：6.04； 五年影响因子：6.485 ）

ISSN：

年卷期： 2021 年 14 卷 1 期

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收录情况： SCI

摘要： Background Humicola insolens is a filamentous fungus with high potential of producing neutral and heat- and alkali-resistant cellulase. However, the genetic engineering tools, particularly the genome-editing tool, are scarce, hindering the study of cellulase expression regulation in this organism. Results Herein, a CRISPR/Cas9 genome-editing system was established in H. insolens based on a hybrid 5S rRNA-tRNA(Gly) promoter. This system is superior to the HDV (hepatitis delta virus) system in genome editing, allowing highly efficient single gene destruction in H. insolens with rates of deletion up to 84.1% (37/44). With this system, a putative pigment synthesis gene pks and the transcription factor xyr1 gene were disrupted with high efficiency. Moreover, the extracellular protein concentration and cellulase activity largely decreased when xyr1 was deleted, demonstrating for the first time that Xyr1 plays an important role in cellulase expression regulation. Conclusions The established CRISPR/Cas9 system is a powerful genetic operation tool for H. insolens, which will accelerate studies on the regulation mechanism of cellulase expression and engineering of H. insolens for higher cellulase production.

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