Abstract
AbstractCandida albicansis 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 ofC. albicansis 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 inC. 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 inC. 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 bothC. albicansand other diploid organisms with strong homologous recombination machinery.
Publisher
Cold Spring Harbor Laboratory