Candida species are opportunistic fungal pathogens that can cause life-threatening infections, particularly in the absence of competition or in immunocompromised individuals. The rise in fungal infections, driven by an increasing at-risk population and climate change, has been accompanied by a surge in antifungal resistance due to widespread antifungal use and the limited availability of antifungal drug classes. To effectively combat antifungal-resistant Candida species and identify new drug targets, a deeper understanding of these pathogens at the genetic level is essential. C. albicans, the most common cause of invasive candidiasis, has become a model organism for studying fungal pathogens due to its similarities to the model yeast Saccharomyces cerevisiae. However, only one-third of the C. albicans genome has been characterized, which hinders the discovery of new antifungal drugs. High-throughput genetic knockout techniques utilizing CRISPR-Cas guide RNA libraries have emerged as powerful tools for functional genomics and rapid drug target identification. Although recent advancements in CRISPR-Cas technologies have greatly facilitated genetic manipulation of C. albicans, high-throughput CRISPR screens in C. albicans have been challenging due to technical barriers including low transformation efficiency, the need for repair templates, and the absence of a plasmid system. Here we present CRISPR-GRIT, a modular repair template and guide RNA fusion design to overcome these technical barriers, paving the way for high-throughput CRISPR screens in C. albicans.
