Advancing functional genetics through Agrobacterium-mediated insertional mutagenesis and CRISPR/Cas9 in the commensal and pathogenic yeast Malassezia furfur

Malassezia encompasses a monophyletic group of basidiomycetous yeasts naturally found on the skin of humans and other animals. Malassezia species have lost genes for lipid biosynthesis, and are therefore lipid-dependent and difficult to manipulate under laboratory conditions. In this study we applied a recently-developed Agrobacterium tumefaciens-mediated transformation protocol to perform T-DNA random insertional mutagenesis in Malassezia furfur. A total of 767 transformants were screened after exposure to 10 different stresses, and the 19 mutants that exhibited a phenotype different from the wild type were further characterized. The majority of these strains had single T-DNA insertions, which were identified within the open reading frames of genes, within untranslated regions, and in intergenic regions. Some T-DNA insertions generated chromosomal rearrangements, and others could not be characterized. To validate the findings of the forward genetic screen, a novel CRISPR/Cas9 system was developed to generate targeted deletion mutants for 2 genes identified in the screen: CDC55 and PDR10. This system is based on co-transformation of M. furfur mediated by A. tumefaciens to deliver both a CAS9-gRNA construct that induces double-strand DNA breaks, and a gene replacement allele that serves as a homology directed repair template. Targeted deletion mutants for both CDC55 and PDR10 were readily generated with this method. This study demonstrates the feasibility and reliability of A. tumefaciens-mediated transformation to aid in the identification of gene functions in M. furfur through both insertional mutagenesis and CRISPR/Cas9-mediated targeted gene deletion.