Ectopic DNA integration and marker-free CRISPR/Cas9 strategies for the halotolerant black yeastHortaea werneckii

Abstract

Hortaea werneckii 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. H. werneckii alternates between fission and budding cell division. This cell division cycle is regulated by cell density and the salt concentration in their media. H. werneckii have been extensively studied due to their ability to grow in high salinity environments and unconventional cell biology. Nonetheless, 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 H. werneckii using PEG-CaCl2 mediated protoplast transformation. This approach relies on a selectable marker (hygromycin B) to select for successful integration of the genetic construct. The same construct was used to express cytosolic green fluorescent protein. Finally, we also developed a marker-free CRISPR/Cas9 protocol for targeted gene deletion. These transformation strategies will allow testing hypotheses related to H. werneckii cell biology and physiology.