The expanding CRISPR toolbox for natural product discovery and engineering in filamentous fungi.

Covering: up to May 2022Fungal genetics has transformed natural product research by enabling the elucidation of cryptic metabolites and biosynthetic steps. The enhanced capability to add, subtract, modulate, and rewrite genes via CRISPR/Cas technologies has opened up avenues for the manipulation of biosynthetic gene clusters across diverse filamentous fungi. This review discusses the innovative and diverse strategies for fungal natural product discovery and engineering made possible by CRISPR/Cas-based tools. We also provide a guide into multiple angles of CRISPR/Cas experiment design, and discuss current gaps in genetic tool development for filamentous fungi and the promising opportunities for natural product research.

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.