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

Area of Science:

• Microbiology
• Synthetic Biology
• Molecular Genetics

Background:

• Debaryomyces hansenii is a halophilic and osmotolerant yeast with significant potential as a cell factory.
• Current genetic tools for D. hansenii are limited, especially for prototrophic strains, due to reliance on auxotrophic markers and challenges with the NHEJ repair pathway.
• Precise gene targeting is crucial for harnessing D. hansenii's full potential but is hindered by existing genetic manipulation methods.

Purpose of the Study:

• To develop a novel, efficient, and versatile gene editing system for prototrophic strains of Debaryomyces hansenii.
• To overcome the limitations of auxotrophic markers and the non-homologous end-joining (NHEJ) DNA repair mechanism in D. hansenii.
• To create a toolset enabling multiplex gene engineering and precise genetic modifications, including point mutations and gene deletions.

Main Methods:

• Development of a plasmid-based CRISPRCUG/Cas9 system utilizing a dominant marker for easy vector assembly.
• Construction of NHEJ-deficient D. hansenii strains to facilitate precise gene editing.
• Utilization of 90-nt single-stranded DNA oligonucleotides for efficient direct repair of Cas9-induced DNA breaks.

Main Results:

• A novel CRISPRCUG/Cas9 toolset for efficient gene editing in prototrophic D. hansenii strains was successfully developed.
• The system supports multiplex gene engineering, enabling simultaneous editing of multiple genes.
• NHEJ-deficient strains and the use of ssDNA oligonucleotides achieved 100% efficiency for precise point mutations and gene deletions.

Conclusions:

• The developed CRISPRCUG/Cas9 tools significantly advance research capabilities in D. hansenii, a promising yeast for cell factory applications.
• These tools facilitate precise genetic modifications, overcoming previous limitations in prototrophic strains.
• The methodology is adaptable for gene editing in other non-conventional yeast species, particularly within the CUG clade.