Abstract The overexpression of genes frequently arises in Nakeseomyces (formerly Candida) glabrata via 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 in N.