CRISPR-Mediated Activation of Biosynthetic Gene Clusters for Bioactive Molecule Discovery in Filamentous Fungi

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 BGCs 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, multigene 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.