Dual sgRNA-directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system

Ganoderma lucidum is an important medicinal mushroom in traditional Chinese medicine. However, the lack of adequate genetic tools has hindered molecular genetic research in and the genetic modification of this species. Here, we report that the presence of an intron is necessary for the efficient expression of the heterologous phosphinothricin-resistance and green fluorescent protein genes in G. lucidum. Moreover, we improved the CRISPR/Cas9-mediated gene disruption frequency in G. lucidum by adding an intron upstream of the Cas9 gene. Our results showed that the disruption frequency of the orotidine 5'-monophosphate decarboxylase gene (ura3) in transformants containing the glyceraldehyde-3-phosphate dehydrogenase gene intron in the Cas9 plasmid is 14-18 in 10⁷ protoplasts, which is 10.6 times higher than that in transformants without any intron sequence. Furthermore, genomic fragment deletions in the ura3 and GL17624 genes were achieved via a dual sgRNA-directed CRISPR/Cas9 system in G. lucidum. We achieved a ura3 deletion frequency of 36.7% in G. lucidum. The developed method provides a powerful platform to generate gene deletion mutants and will facilitate functional genomic studies in G. lucidum.