The emergence of virulent, resistant, and rapidly evolving fungal pathogens poses a significant threat to public health, agriculture, and the environment. Standard small-molecule intervention targeting cellular processes may be effective but requires long development times and is prone to antibiotic resistance. To overcome the current limitations of antibiotic development and treatment, this study harnesses CRISPR-Cas systems as antifungals by capitalizing on their adaptability, specificity, and efficiency in target design. While conventional CRISPR-Cas antimicrobials rely on the lethality of DNA double-strand breaks (DSBs), this is potentially less effective in fungi due to robust eukaryotic DNA repair machinery. Here, we present the novel design strategy of cotargeting essential genes with DNA repair defensive genes that remove the fungi’s ability to repair the DSB sites of essential genes. By evaluating this design on the model yeast Saccharomyces cerevisiae, we demonstrated that essential and defensive gene cotargeting is more effective than either essential or defensive gene targeting alone. Cotargeting essential genes with RAD52, involved in homologous recombination, proved to be the most synergistic combination tested. Fast growth kinetics of S. cerevisiae led to resistance to CRISPR-Cas antifungals where escapee populations predominantly carried mutations in defensive genes and guide RNA sequences. Here we present current efforts to investigate the applicability of this approach in pathogenic Candida species.
