C. J. Cotter, C. T. Trinh
Drug-resistant fungal pathogens pose a growing public health threat, causing millions of infections and deaths annually. Limited antifungal drug classes and rising resistance highlight the urgent need for novel therapies. CRISPR-Cas systems offer sequence-specific antimicrobial potential, but their efficacy is influenced by organism-specific DNA repair outcomes. Here, we demonstrate that in Candida albicans, which predominantly relies on homology-directed repair (HDR), both repair template availability and DNA repair enzyme activity critically determine Cas9-induced lethality. By providing Trojan Horse donor DNA repair templates when targeting essential and DNA repair genes, we show that Cas9 lethality can be selectively tuned. Furthermore, multiplexed gRNA targeting to modulate DNA repair capacity reveals strong synergistic interactions when co-targeting HDR components, which is corroborated by enhanced killing in HDR-compromised strains. These results establish DNA repair as a programmable determinant of CRISPR-Cas antifungal activity and provide a mechanistic framework for combinatorial targeting strategies, advancing the development of CRISPR-Cas antifungals.