Genetic Transformation of Candida auris via Homology-Directed Repair Using a Standard Lithium Acetate Protocol

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• Reverse genetics is crucial for understanding gene function in non-model organisms.
• Developing genetic manipulation techniques for emerging pathogens like Candida auris is challenging.
• Candida auris is a significant human pathogen requiring effective research tools.

Purpose of the Study:

• To establish a reliable genetic transformation protocol for Candida auris.
• To enable gene function studies in Candida auris using reverse genetics.
• To facilitate the characterization of virulence factors and drug resistance mechanisms.

Main Methods:

• Developed a protocol for DNA construct generation using fusion PCR for gene deletion with drug resistance markers.
• Optimized chemical competence and transformation procedures for Candida auris.
• Utilized long flanking homologous sequences (>1 kb) for homology-directed repair integration.
• Confirmed successful DNA integration via PCR analysis.

Main Results:

• Successfully established a genetic transformation protocol for Candida auris.
• Demonstrated the ability to perform gene deletions using homology-directed repair.
• The protocol allows for targeted integration of DNA constructs into the Candida auris genome.
• PCR confirmed the accurate integration of constructs.

Conclusions:

• The described protocol provides a robust method for genetic manipulation in Candida auris.
• This tool will accelerate research into Candida auris pathogenesis and antifungal resistance.
• The strategy is adaptable for various genetic modifications, including promoter exchanges and protein tagging.