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bioRxiv · 10.1101/2020.01.12.903286

CRISPR-mediated activation of biosynthetic gene clusters for bioactive molecule discovery in filamentous fungi

Roux, I.·Woodcraft, C.·Hu, J.·Wolters, R.·Gilchrist, C. L. M.·Chooi, Y.-H.
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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 BGC 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, multi-gene 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.Competing Interest StatementThe authors have declared no competing interest.View Full Text

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Roux, I., Woodcraft, C., Hu, J., Wolters, R., Gilchrist, C. L. M., Chooi, Y.-H.. 2020-01-14. CRISPR-mediated activation of biosynthetic gene clusters for bioactive molecule discovery in filamentous fungi. https://doi.org/10.1101/2020.01.12.903286

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