CRISPR/Cas9-Mediated Genome Engineering Reveals the Contribution of the 26S Proteasome to the Extremophilic Nature of the Yeast Debaryomyces hansenii

Abstract

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.

A CRISPR/Cas9 method facilitates efficient oligo-mediated gene editing in Debaryomyces hansenii

“…The highest efficiency (∼85%) was achieved with pDIV510 that features a heterologous Cl_TDH3p promoter driving Cas9 CUG expression ( Figure 1C ). However, transformants obtained with this plasmid displayed an increased heterogeneity in colony size, which potentially could be due to high toxic levels of the Cas9 nuclease ( 16 ). Together, the results above strongly indicate that our CRISPR CUG technology works robustly in D. hansenii .…”

“…The slow development of genetic tools for D. hansenii has been affected by several factors. For example, efficient transformation protocols were not available until recent years ( 16 , 17 ). There are only few known circular plasmids that can propagate in D. hansenii ; however, they rely on auxotrophic markers and are only applicable for specific laboratory strains ( 18–20 ).…”

“…This fact further complicates the heterologous expression in this yeast as all genes of interest (GOIs) need to be codon-optimized to exclude CUG codons. Finally, but very importantly, genetic engineering of this yeast has been very limited as the non-homologous end-joining (NHEJ) pathway is preferred for integrating DNA into chromosomes ( 16 ). This fact makes it very difficult to introduce precise genome alterations via homology-directed gene targeting and gene editing in this species ( 16 , 18 ).…”

“…Finally, but very importantly, genetic engineering of this yeast has been very limited as the non-homologous end-joining (NHEJ) pathway is preferred for integrating DNA into chromosomes ( 16 ). This fact makes it very difficult to introduce precise genome alterations via homology-directed gene targeting and gene editing in this species ( 16 , 18 ). Discovery of CRISPR/Cas9 and its successful application have revolutionized the genetic engineering and synthetic biology fields in many species ( 22 ).…”

“…This technology has been successfully adapted for genome engineering of several NCYs ( 23 ). Indeed, a recent study has recently demonstrated the application of CRISPR/Cas9 in D. hansenii ; however, it was mainly used for gene inactivation via flawed NHEJ repair, while attempts to perform homologous recombination (HR)-directed edits had low success ( 16 ). Thus, there is a high need for an improved genetic toolbox for D. hansenii that allows for swift and robust gene targeting and gene editing.…”