AbstractMalassezia are a monophyletic phylum of 18 species of commensal/ pathogenic yeasts that are prominent in the mammalian mycobiome, and recently linked to risk of Crohn’s Disease. Malassezia are challenging to study...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 transfer (T)-DNA random insertional mutagenesis in Malassezia furfur. A total of 767 transformants were screened for sensitivity to 10 different stresses, and 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 open reading frames of genes, untranslated regions, and intergenic regions. Some T-DNA insertions generated chromosomal rearrangements while others could not be characterized. To validate the findings of our forward genetic screen, a novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was developed to generate targeted deletion mutants for two genes identified in the screen: CDC55 and PDR10. This system is based on cotransformation 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.