Advancing Functional Genetics Through -Mediated Insertional Mutagenesis and CRISPR/Cas9 in the Commensal and Pathogenic Yeast .

encompasses a monophyletic group of basidiomycetous yeasts naturally found on the skin of humans and other animals. 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 -mediated transformation protocol to perform transfer (T)-DNA random insertional mutagenesis in 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: and This system is based on cotransformation of mediated by , to deliver both a -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 and were readily generated with this method. This study demonstrates the feasibility and reliability of -mediated transformation to aid in the identification of gene functions in , through both insertional mutagenesis and CRISPR/Cas9-mediated targeted gene deletion.