| Summary: | Marine-derived fungi are emerging as attractive producers of structurally novel secondary metabolites with diverse bioactivities. However, the lack of efficient genetic tools limits the discovery of novel compounds and the elucidation of biosynthesis mechanisms. Here, we firstly established an effective PEG-mediated chemical transformation system for protoplasts in two marine-derived fungi, <i>Spiromastix</i> sp. SCSIO F190 and <i>Aspergillus</i> sp. SCSIO SX7S7. Next, we developed a simple and versatile CRISPR-Cas9-based gene disruption strategy by transforming a target fungus with a single plasmid. We found that the transformation with a circular plasmid encoding <i>cas9</i>, a single-guide RNA (sgRNA), and a selectable marker resulted in a high frequency of targeted and insertional gene mutations in both marine-derived fungal strains. In addition, the histone deacetylase gene <i>rpd3</i> was mutated using the established CRISPR-Cas9 system, thereby activating novel secondary metabolites that were not produced in the wild-type strain. Taken together, a versatile CRISPR-Cas9-based gene disruption method was established, which will promote the discovery of novel natural products and further biological studies.
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