Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Shanxi Medical University, Taiyuan 030001, Shanxi, China; Shanxi Academy of Advanced Research and Innovation, Taiyuan 030032, Shanxi, China.
Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Shanxi Medical University, Taiyuan 030001, Shanxi, China.
Int J Biol Macromol. 2024 Nov;279(Pt 4):135339. doi: 10.1016/j.ijbiomac.2024.135339. Epub 2024 Sep 6.
The CRISPR/Cas9 genome editing tool has been extensively utilized in filamentous fungi, including Trichoderma reesei. However, most existing systems employ constitutive promoters for the expression of Cas9 protein within the cells or directly introduce Cas9 protein into the cells, which often leads to continuous expression of Cas9 resulting in undesired phenotypes or increased operational cost. In this study, we identified a quinic acid (QA)-induced qai5 promoter and employed it to express Cas9, thereby establishing an inducible genome editing system in T. reesei. By utilizing this system, we successfully edited the ypr1 gene and the silent gene cluster involved in ilicicolin H synthesis using donor DNA labeling 41-bp homology arm (HA), resulting in an editing efficiency ranging from 29.2 % to 46.7 %. Consequently, biosynthesis of ilicicolin H was achieved in T. reesei. To summarize, this study presents a novel form of CRISPR/Cas9 genome editing system that enables efficient and controllable genetic modifications in T. reesei.
CRISPR/Cas9 基因组编辑工具已被广泛应用于丝状真菌，包括里氏木霉。然而，大多数现有的系统在细胞内使用组成型启动子表达 Cas9 蛋白，或者直接将 Cas9 蛋白导入细胞，这通常导致 Cas9 的持续表达，从而导致不理想的表型或增加运营成本。在本研究中，我们鉴定了一种奎尼酸 (QA) 诱导的 qai5 启动子，并利用它来表达 Cas9，从而在里氏木霉中建立了一个诱导型基因组编辑系统。利用该系统，我们成功地编辑了 ypr1 基因和参与伊利西醇 H 合成的沉默基因簇，使用供体 DNA 标记 41-bp 同源臂 (HA)，编辑效率范围为 29.2%至 46.7%。因此，在里氏木霉中实现了伊利西醇 H 的生物合成。总之，本研究提出了一种新型的 CRISPR/Cas9 基因组编辑系统，能够在里氏木霉中进行高效和可控的遗传修饰。
