A Genome-Wide CRISPR Screening Platform in Diploid Candida Albicans to Uncover Mechanisms of Antifungal Resistance - Multidrug-resistant fungal pathogens pose a significant threat to public health. Some Candida pathogens contribute to over one billion infections and approximately 1.5 million human deaths globally each year. Among these, the well- characterized C. albicans causes systemic infections with mortality rates of up to 72%, and is increasingly resistant to current antifungal drugs. Advancing our understanding of fungal molecular biology and gene-function relationships in Candida species is essential to elucidate the molecular mechanisms of antifungal resistance and inform strategies for combating multidrug-resistant fungi. However, the relationships between genes and functions conferring fungal robustness, including multidrug-resistant phenotypes, are complex and remain largely unexplored in Candida species, even in C. albicans, where approximately one-third of the proteome has only been annotated through comparative genomics. Functional genomics approaches such as gene knockout libraries via transposon mutagenesis and homologous recombination, have been developed for C. albicans over several decades, but these methods offer limited genome coverage. Major obstacles include complex polyploid genomes, low transformation efficiency, absence of autonomously replicating plasmids, and the need for multiplexed genome editing to dissect traits governed by multiple cellular processes. The goal of this exploratory project is to overcome key limitations in high-throughput functional genomics by developing tools for efficient DNA transformation and CRISPR-based genome editing in C. albicans, and applying these tools to perform genome-wide CRISPR screening to identify genes and cellular processes critical for antifungal resistance. This work builds on our recent progress in developing synthetic biology tools for diploid C. albicans, which may be extended to other Candida species. We improved chemical transformation efficiency by over two orders of magnitude, from 102 to 104 cfu/g DNA, by modulating cell stress tolerance. We also developed synthetic guide RNAs (gRNAs) with integrated repair templates, referred to as CRISPR- GRIT, which comprise tRNA, repair template, seed sequence, and scaffold, enabling precise, multiplexable genome editing without the need for large donor templates. To achieve our goal, we propose two exploratory aims. Aim 1 will develop foundational synthetic biology toolboxes for high-throughput, genome-wide functional genomics in diploid C. albicans. We will engineer autonomously replicating linear plasmids, coupled with a modular CRISPR-GRIT design, for precise genome editing. In addition, we will systematically characterize environmental variables (e.g., chemical, pH, temperature) and genetic variables (e.g., replicating plasmids) to optimize transformation conditions and achieve >106 cfu/g, a critical threshold for genome-wide CRISPR screening. Aim 2 will construct a pooled CRISPR library targeting all genes in C. albicans and initiate screening to identify genetic determinants of antifungal resistance. This high-risk, high-reward project will develop innovative synthetic biology tools for high-throughput functional genomics in difficult- to-manipulate fungi, advancing our fundamental knowledge of gene functions and biological processes underlying important phenotypes such as antifungal resistance, and laying the foundation for antifungal therapeutic development.
