Potency of CRISPR-Cas Antifungals Is Enhanced by Co-targeting DNA Repair and Growth Regulatory Machinery at the Genetic Level

Fungal pathogens are virulent and resistant to antibiotic treatment. Due to their adaptability, specificity, and efficiency, CRISPR-Cas systems can be harnessed for neutralizing these fungal pathogens. However, the conventional design of CRISPR-Cas antimicrobials, based on induction of DNA double-strand-breaks (DSBs), is potentially less effective in fungi due to robust eukaryotic DNA repair machinery. Here, we report a novel design principle to formulate more effective CRISPR-Cas antifungals by co-targeting 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 fungus Saccharomyces cerevisiae , we demonstrated that essential and defensive gene co-targeting is more effective than either essential or defensive gene targeting alone. The top-performing CRISPR-Cas antifungals performed as effectively as the antibiotic Geneticin. Fast growth kinetics of S. cerevisiae induced resistance to CRISPR-Cas antifungals where genetic mutations mostly occurred in defensive genes and guide RNA sequences. Significance The emergence of virulent, resistant, and rapidly evolving fungal pathogens poses a significant threat to public health, agriculture, and the environment. Targeting cellular processes with standard small-molecule intervention may be effective but requires long development times and is prone to antibiotic resistance. To overcome the current limitation of antibiotic development and treatment, this study harnesses CRISPR-Cas systems as antifungals by capitalizing on their adaptability, specificity, and efficiency in target design. Simultaneous co-targeting of both essential and defensive genes is shown to be a novel design principle for formulating effective CRISPR-Cas antimicrobials that can be rapidly tuned to adapt to inevitable escapee events.