CRISPR-GRIT: Guide-RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen Candida albicans

Candida albicans is an opportunistic fungal pathogen that causes severe infections in immunocompromised individuals. Overuse of antifungals coupled with climate change has led to the rapid emergence of antifungal resistance. Thus, there is an urgent need to understand fungal pathogen genetics to develop new antifungal strategies. Genetic manipulation of C. albicans is encumbered by its diploid chromosomes which require editing both alleles for elucidating gene function. Even though recent development of CRISPR-Cas systems has facilitated genome editing in C. albicans, large-scale functional genomic studies are still hindered by the necessity of co-transforming repair templates for homozygous knockouts. Here, we present CRISPR-GRIT, a repair template-integrated guide RNA design for expedited gene knockouts in C. albicans, and demonstrate its utility for multiplexed editing. We envision that this method can be employed for high-throughput library screens and identification of synthetic lethal pairs in both C. albicans and other diploid organisms with strong homologous recombination machinery.