Pooled CRISPRi and CRISPRa strain library build for Candida albicans v1

This protocol is for constructing and screening pooled CRISPR interference and activation (CRISPRi/a) libraries in C. albicans. This workflow integrates sgRNA design, pooled cloning, bacterial and fungal library generation, competitive growth assays, and next-generation sequencing-based quantification of strain abundance. A key feature of this CRISPRi/a system is a regulatable dCas9 system that enables temporal control of gene perturbation, allowing interrogation of essential genes. The approach is modular and scalable, supporting the generation of fungal mutant pools ranging from targeted gene sets to genome-scale libraries, and is readily transferable across diverse C. albicans strain backgrounds, including clinical isolates. Pooled libraries can be generated within approximately one week, and can be screened in diverse environmental conditions to quantify gene fitness effects, enabling systematic identification and characterization of gene function in this critical human fungal pathogen.