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

<i>Candida albicans,</i> an opportunistic fungal pathogen, causes severe infections in immunocompromised individuals. Limited classes and overuse of current antifungals have 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 <i>C. albicans</i> is encumbered by its diploid chromosomes requiring editing both alleles to elucidate gene function. Although the recent development of CRISPR-Cas systems has facilitated genome editing in <i>C. albicans</i>, large-scale and multiplexed functional genomic studies are still hindered by the necessity of cotransforming repair templates for homozygous knockouts. Here, we present CRISPR-GRIT (<u>G</u>uide <u>R</u>NAs with <u>I</u>ntegrated Repair <u>T</u>emplates), a repair template-integrated guide RNA design for expedited gene knockouts and multiplexed gene editing in <i>C. albicans</i>. We envision that this method can be used for high-throughput library screens and identification of synthetic lethal pairs in both <i>C. albicans</i> and other diploid organisms with strong homologous recombination machinery.