Functional genetic characterization of stress tolerance and biofilm formation inNakaseomyces glabratavia a novel CRISPR activation system

Abstract

The overexpression of genes frequently arises inNakaseomyces(formerlyCandida)glabratavia gain-of-function mutations, gene duplication or aneuploidies, with important consequences on pathogenesis traits and antifungal drug resistance. This highlights the need to develop specific genetic tools to mimic and study genetic amplification in this important fungal pathogen. Here, we report the development, validation, and applications of the first CRISPR activation (CRISPRa) system inN. glabratafor targeted genetic overexpression. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression inN. glabrata, and further assess optimal guide RNA targeting for robust overexpression. We demonstrate the applications of CRISPRa to overexpress genes involved in fungal pathogenesis and drug resistance, and detect corresponding phenotypic alterations in these key traits, including the characterization of novel phenotypes. Finally, we capture strain variation using our CRISPRa system in two commonly usedN. glabratagenetic backgrounds. Together, this tool will expand our capacity for functional genetic overexpression in this pathogen, with numerous possibilities for future applications.