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

ACS Synth Biol. 2020 Jul 17;9(7):1843-1854. doi: 10.1021/acssynbio.0c00197. Epub 2020 Jun 26.

Abstract

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.

Keywords: Aspergillus nidulans; CRISPRa; Cas12a; biosynthetic gene clusters; filamentous fungi; secondary metabolites.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspergillus nidulans / genetics*
  • Bacterial Proteins / genetics
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Culture Media
  • Drug Discovery / methods*
  • Endodeoxyribonucleases / genetics
  • Firmicutes / enzymology
  • Genes, Fungal*
  • Multigene Family*
  • Peptide Synthases / genetics
  • Promoter Regions, Genetic
  • RNA, Guide / genetics
  • Streptococcus pyogenes / enzymology
  • Temperature
  • Transcription, Genetic / genetics
  • Transcriptional Activation*

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • Culture Media
  • RNA, Guide
  • CRISPR-Associated Protein 9
  • Cas12a protein
  • Endodeoxyribonucleases
  • Peptide Synthases
  • non-ribosomal peptide synthase