TY - JOUR T1 - CRISPR-mediated activation of biosynthetic gene clusters for bioactive molecule discovery in filamentous fungi JF - bioRxiv DO - 10.1101/2020.01.12.903286 SP - 2020.01.12.903286 AU - Indra Roux AU - Clara Woodcraft AU - Jinyu Hu AU - Rebecca Wolters AU - Cameron L.M. Gilchrist AU - Yit-Heng Chooi Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/05/20/2020.01.12.903286.abstract N2 - 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 dLbCas12aD156R-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.Competing Interest StatementThe authors have declared no competing interest. ER -