Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii : ectopic DNA integration and marker-free CRISPR/Cas9 transformation

Abstract

Hortaea werneckii is a remarkable yeast capable of growing in high salt concentration, and its cell division cycle alternates between fission-like and budding. For these unique attributes, H. werneckii has gathered interest in research programs studying extremophile fungi and cell division. Most of our understanding of H. werneckii biology comes from genomic analyses, the usage of drugs to target a particular pathway, or the heterologous expression of its genes in S. cerevisiae . Nonetheless, H. werneckii has remained genetically intractable. Here, we report on two strategies to transform H. werneckii : ectopic integration of a plasmid and gene deletion using CRISPR/Cas9. These approaches will be fundamental to expanding the experimental techniques available to study H. werneckii , including live-cell imaging of cellular processes and reverse genetic approaches.