The marine yeast Debaryomyces hansenii is of high importance in the food, chemical, and medical industries. D. hansenii is also a popular model for studying molecular mechanisms of halo-and osmotolerance. The absence of genome editing technologies hampers D. hansenii research and limits its biotechnological application. We developed novel and efficient single-and dual-guide CRISPR systems for markerless genome editing of D. hansenii. The single-guide system allows highefficiency (up to 95%) mutation of genes or regulatory elements. The dual-guide system is applicable for efficient deletion of genomic loci. We used these tools to study transcriptional regulation of the 26S proteasome, an ATP-dependent protease complex whose proper function is vital for all cells and organisms. We developed a genetic approach to control the activity of the 26S proteasome by deregulation of its essential subunits. The mutant strains were sensitive to geno-and proteotoxic stresses as well as high salinity and osmolarity, suggesting a contribution of the proteasome to the extremophilic properties of D. hansenii. The developed CRISPR systems allow efficient D. hansenii genome engineering, providing a genetic way to control proteasome activity, and should advance applications of this yeast.