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 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..
7Accessing the full biosynthetic potential encoded in the genomes of fungi is limited by the low 8 expression of many biosynthetic gene clusters (BGCs) under standard culture conditions. In 9 this work, we develop a fungal CRISPR activation (CRISPRa) system for targeted upregulation 10 of biosynthetic genes, which could accelerate the emerging genomics-driven approach to 11 bioactive secondary metabolite discovery. We construct a fungal CRISPR/dLbCas12a-VPR 12 system and demonstrate activation of a fluorescent reporter in Aspergillus nidulans. Then, we 13 target the native nonribosomal peptide synthetase-like (NRPS-like) gene micA in both 14 chromosomal and episomal contexts, achieving increases in production of the compound 15 microperfuranone. Finally, multi-gene CRISPRa leads to the discovery of the mic cluster 16 product as dehydromicroperfuranone. Additionally, we demonstrate the utility of the variant 17 LbCas12a D156R -VPR for CRISPRa at lower culture temperatures. This is the first 18 demonstration of CRISPRa in filamentous fungi, providing a framework for CRISPR-mediated 19 transcriptional activation of fungal BGCs. 20 activation (CRISPRa)-mediated approach for BGC activation in filamentous fungi (Fig. 1a). In 49 CRISPRa systems, DNase-deactivated RNA-guided CRISPR/dCas ribonucleoprotein 50 complexes linked to activation effectors are targeted to gene regulatory regions to increase 51 gene expression [17][18][19] . Taking advantage of the streamlined CRISPR RNA (crRNA) cloning 52 and multiplexing capabilities, CRISPRa of BGCs has the potential to greatly accelerate fungal 53 genome mining. CRISPRa has already been used to tune the expression of biosynthetic 54 pathways 20,21 , including in ascomycetous yeasts 22,23 . However, to our knowledge, CRISPRa 55has not yet been demonstrated in filamentous fungi. 56In this work, we develop a suite of fungal CRISPRa vectors based on both dLbCas12a from 57Lachnospiraceae bacterium Cas12a (previously known as Cpf1) 19 , and dSpCas9 from 58Streptococcus pyogenes fused to the tripartite VPR activator 18 , and test them in A. nidulans. 59We further explore the application of our CRISPR/dLbCas12a-VPR system for fungal BGC 60 activation as a tool to fuel bioactive molecule discovery. 61
Results
62
Construction and testing of fungal CRISPRa systems 63To develop a CRISPRa system for filamentous fungi, we constructed and tested 64 CRISPR/dLbCas12a-VPR-and CRISPR/dSpCas9-VPR-based systems in the model 65 organism and chassis A. nidulans. To evaluate alternative strategies for expressing either 66 3 dCas effector, we created parent strains with a chromosomally integrated dCas-VPR 67 expression cassette and compared their performance with entirely AMA1-episomally encoded 68 systems. The AMA1 sequence acts as an extrachromosomal vector replicator and confers 69 increased transformation frequency in several filamentous fungi species 24 . AMA1-bearing 70 vectors are found at multiple copies per nucleus although their genetic stability has been 71 reported to be limited under non...
Burnettiene A is a novel cytotoxic tridecaketide decalin polyketide from Aspergillus burnettii. Its biosynthesis was elucidated by heterologous expression in fungi.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.