HyperdCas12a-based multiplexed genetic regulation in Candida albicans.

Area of Science:

• Microbiology
• Mycology
• Molecular Biology

Background:

• Complex microbial phenotypes arise from intricate gene regulatory networks.
• Current reverse genetics in microbial pathogens often rely on single-gene studies due to limited genetic tools.
• Advancements in CRISPR-Cas platforms are crucial for scalable microbial functional genomics.

Purpose of the Study:

• To introduce and validate a hyper-efficient CRISPR-Cas12a system for multiplexed gene activation and repression in Candida albicans.
• To explore the utility of this system for studying gene interactions and drug resistance mechanisms.
• To establish a novel tool for functional genomics in human fungal pathogens.

Main Methods:

• Translating the hyperdCas12a variant to Candida albicans.
• Developing CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) tools.
• Profiling the efficiency and tunability of gene modulation.
• Generating combinatorial gene perturbations in drug efflux and ergosterol biosynthesis pathways.

Main Results:

• Demonstrated highly efficient, inducible, and multiplexed gene activation and repression in Candida albicans.
• Achieved tunable levels of target gene modulation using the hyperdCas12a system.
• Identified gene redundancies and synergistic interactions in drug resistance circuitry through combinatorial perturbations.
• Established the first CRISPR-Cas12 system application in a human fungal pathogen.

Conclusions:

• The hyperdCas12a platform provides an efficient method for generating combinatorial mutants in Candida albicans.
• This system enables a deeper mechanistic understanding of genetic interactions underlying fungal phenotypes and drug resistance.
• The enhanced CRISPR-Cas12a activity in fungi suggests potential translation to other microbial systems for genetic interaction studies.