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

ACS Synthetic Biology
Indra RouxYit-Heng Chooi

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.

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Citations

Jan 15, 2021·Scientific Reports·László MózsikArnold J M Driessen
Jan 23, 2021·Microbial Cell Factories·Michael Dare AsemoloyeLorenzo Pecoraro
Mar 2, 2021·Transgenic Research·Teng-Kuei Huang, Holger Puchta
May 16, 2021·Fungal Genetics and Biology : FG & B·Nicolau SbarainiCharley Christian Staats
Jan 12, 2021·Briefings in Bioinformatics·Hui PengJinyan Li
Nov 3, 2021·ACS Synthetic Biology·László MózsikArnold J M Driessen

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