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

Halophilic and osmotolerant yeast has a high potential for cell factory applications due to its resistance to harsh environmental factors and compatibility with a wide substrate range. However, currently available genetic techniques do not allow the full potential of as a cell factory to be harnessed. Moreover, most of the currently available tools rely on the use of auxotrophic markers that are not suitable in wild-type prototrophic strains. In addition, the preferred non-homologous end-joining (NHEJ) DNA damage repair mechanism poses further challenges when precise gene targeting is required. In this study, we present a novel plasmid-based CRISPR/Cas9 method for easy and efficient gene editing of the prototrophic strains of . Our toolset design is based on a dominant marker and facilitates quick assembly of the vectors expressing Cas9 and single or multiple single-guide RNAs (sgRNAs) that provide the possibility for multiplex gene engineering even in prototrophic strains. Moreover, we have constructed NHEJ-deficient that enable our CRISPR/Cas9 tools to support the highly efficient introduction of point mutations and single/double gene deletions. Importantly, we also demonstrate that 90-nt single-stranded DNA oligonucleotides are sufficient for direct repair of DNA breaks induced by sgRNA-Cas9, resulting in precise edits reaching 100% efficiencies. In conclusion, tools developed in this study will greatly advance basic and applied research in In addition, we envision that our tools can be rapidly adapted for gene editing of other non-conventional yeast species including the ones belonging to the CUG clade.