Abstract
Filamentous fungi represent an invaluable source of pharmaceutically active compounds. The development of versatile methods to genetically manipulate filamentous fungi is of great value for improving the low yields of bioactive metabolites and expanding chemical diversity. The CRISPR-Cas9-based system has become a common platform for genome editing in a variety of organisms. However, recent application of this technology in filamentous fungi is limited to model strains, a versatile method for efficient gene disruption in different fungi is lacking. Here, we investigated the utility of the CRISPR-Cas9 system in a less-studied fungus Nodulisporium sp. (No. 65-12-7-1), and we have developed an efficient CRISPR-Cas9-based gene disruption strategy by simultaneous transformation of in vitro transcriptional gRNA and the linear maker gene cassette into the Cas9-expressing fungi. We found that the linear marker gene cassette could not only allow for selection of transformants, but also significantly enhance the gene disruption efficiency by inserting itself into the Cas9 cut site. Moreover, the above approach also demonstrated its efficiency in two other phylogenetically distinct strains Aspergillus oryzae NSAR1 and Sporormiella minima (No. 40-1-4-1) from two different classes of Ascomycota. These results suggested that a versatile CRISPR-Cas9-based gene disruption method in filamentous fungi was established.
Highlights
Filamentous fungi serve as an invaluable source of biologically active natural products including the most widely used antibacterials penicillin and cephalosporin, the anti-fungals griseofulvin and echinocandin, and statins, which are a class of cholesterol-lowering agents[1]
The applications of CRISPR-Cas[9] technology in filamentous fungi have drawn attention and their validity has been demonstrated in several fungal strains including Pyricularia oryzae[10], A. fumigatus[11], A. oryzae[12], A. niger[13], Trichoderma reesei[14], Talaromyces atroroseus[15], Penicillium chrysogenum[16], Neurospora crassa[17], Ustilago maydis[18], Candida albicans[19], Alternaria alternata[20]
Gene mutagenesis was achieved during repair of the double-stranded breaks (DSBs) induced by CRISPR-Cas[9] through either non-homologous end joining (NHEJ) or HDR10, 12, 15, 17, 26
Summary
Filamentous fungi serve as an invaluable source of biologically active natural products including the most widely used antibacterials penicillin and cephalosporin, the anti-fungals griseofulvin and echinocandin, and statins, which are a class of cholesterol-lowering agents[1]. The applications of CRISPR-Cas[9] technology in filamentous fungi have drawn attention and their validity has been demonstrated in several fungal strains including Pyricularia oryzae[10], A. fumigatus[11], A. oryzae[12], A. niger[13], Trichoderma reesei[14], Talaromyces atroroseus[15], Penicillium chrysogenum[16], Neurospora crassa[17], Ustilago maydis[18], Candida albicans[19], Alternaria alternata[20] Most of these studies are limited to model strains belonging to a relatively small subset of Ascomycota (such as Aspergillus, Penicillium and Neurospora) that have been thoroughly studied and are genetically tractable. Establishment of a genetic manipulation system in this fungus would be of great value to reveal the biosynthetic assembly of demethoxyviridin and its derivatives
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