Accessing the full biosynthetic potential encoded in the genomes of fungi is limited by the low expression of most biosynthetic gene clusters (BGCs) under common laboratory culture conditions. CRISPR-mediated transcriptional activation (CRISPRa) of fungal BGC could accelerate genomics-driven bioactive secondary metabolite discovery. In this work, we established the first CRISPRa system for filamentous fungi. First, we constructed a CRISPR/dLbCas12a-VPR-based system and demonstrated the activation of a fluorescent reporter in Aspergillus nidulans. Then, we targeted the native nonribosomal peptide synthetase-like (NRPS-like) gene micA in both chromosomal and episomal contexts, achieving increased production of the compound microperfuranone. Finally, multi-gene CRISPRa led to the discovery of the mic cluster product as dehydromicroperfuranone. Additionally, we demonstrated the utility of the variant dLbCas12a^D156R-VPR for CRISPRa at room temperature culture conditions. Different aspects that influence the efficiency of CRISPRa in fungi were investigated, providing a framework for the further development of fungal artificial transcription factors based on CRISPR/Cas.

中文翻译：

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CRISPR介导的丝状真菌生物活性分子发现的生物合成基因簇的激活

在常规实验室培养条件下，大多数生物合成基因簇（BGC）的低表达限制了真菌基因组中编码的全部生物合成潜力的获取。真菌BGC的CRISPR介导的转录激活（CRISPRa）可以加速基因组学驱动的生物活性次级代谢产物的发现。在这项工作中，我们建立了第一个用于丝状真菌的CRISPRa系统。首先，我们构建了一个基于CRISPR / dLbCas12a-VPR的系统，并证明了构巢曲霉中荧光报告分子的激活。然后，我们靶向天然的非核糖体肽合成酶样（NRPS样）基因micA在染色体和游离环境中，都能提高化合物微呋喃酮的产量。最终，多基因CRISPRa导致了麦克风簇产品脱氢微呋喃酮的发现。另外，我们展示了变体dLbCas12a ^D156R -VPR在室温培养条件下对CRISPRa的效用。研究了影响CRISPRa在真菌中效率的不同方面，为进一步开发基于CRISPR / Cas的真菌人工转录因子提供了框架。