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/2020/01/14/2020.01.12.903286.abstract N2 - Accessing the full biosynthetic potential encoded in the genomes of fungi is limited by the low expression of many biosynthetic gene clusters (BGCs) under standard culture conditions. In this work, we develop a fungal CRISPR activation (CRISPRa) system for targeted upregulation of biosynthetic genes, which could accelerate the emerging genomics-driven approach to bioactive secondary metabolite discovery. We construct a fungal CRISPR/dLbCas12a-VPR system and demonstrate activation of a fluorescent reporter in Aspergillus nidulans. Then, we target the native nonribosomal peptide synthetase-like (NRPS-like) gene micA in both chromosomal and episomal contexts, achieving increases in production of the compound microperfuranone. Finally, multi-gene CRISPRa leads to the discovery of the mic cluster product as dehydromicroperfuranone. Additionally, we demonstrate the utility of the variant LbCas12aD156R-VPR for CRISPRa at lower culture temperatures. This is the first demonstration of CRISPRa in filamentous fungi, providing a framework for CRISPR-mediated transcriptional activation of fungal BGCs. ER -