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

<i>Hortaea werneckii</i> is a halotolerant black yeast commonly found in hypersaline environments. This yeast is also the causative agent of tinea nigra, a superficial mycosis of the palm of the hand and soles of the feet of humans. In addition to their remarkable halotolerance, this black yeast exhibits an unconventional cell division cycle, alternating between fission and budding cell division. Cell density and the salt concentration in their environment regulate which cell division cycle <i>H. werneckii</i> uses. Although <i>H. werneckii</i> have been extensively studied due to their unique physiology and cell biology, deciphering the underlying mechanisms behind these remarkable phenotypes has been limited due to the lack of genetic tools available. Here, we report a new ectopic integration protocol for <i>H. werneckii</i> using polyethylene glycol-CaCl₂ mediated protoplast transformation. This approach relies on a drug (hygromycin B) resistance gene to select for successful integration of the genetic construct. The same construct was used to express cytosolic green fluorescent protein. Finally, we developed a marker-free CRISPR/Cas9 protocol for targeted gene deletion using the melanin synthesis pathway as a visual reporter of successful transformation. These transformation strategies will allow testing hypotheses related to <i>H. werneckii</i> cell biology and physiology.IMPORTANCE<i>Hortaea werneckii</i> 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, <i>H. werneckii</i> has gathered interest in research programs studying extremophile fungi and cell division. Most of our understanding of <i>H. werneckii</i> biology comes from genomic analyses, the usage of drugs to target a particular pathway, or the heterologous expression of its genes in <i>S. cerevisiae</i>. Nonetheless, <i>H. werneckii</i> has remained genetically intractable. Here, we report on two strategies to transform <i>H. werneckii</i>: ectopic integration of a plasmid and gene deletion using CRISPR/Cas9. These approaches will be fundamental to expanding the experimental techniques available to study <i>H. werneckii</i>, including live-cell imaging of cellular processes and reverse genetic approaches.