期刊
ACS SYNTHETIC BIOLOGY
卷 9, 期 7, 页码 1843-1854出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.0c00197
关键词
CRISPRa; filamentous fungi; Cas12a; Aspergillus nidulans; biosynthetic gene dusters; secondary metabolites
资金
- ARC Future Fellowship [FT160100233]
- UWA Ph.D. Scholarship
- Australian Government Research Training Program scholarships
Accessing the full biosynthetic potential encoded in the genomes of fungi is limited by the low expression of most biosynthetic gene dusters (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 dLbCas12a(D156R)-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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