Design and Generation of a CRISPR Interference System for Genetic Repression and Essential Gene Analysis in the...

Studying life-threatening fungal pathogens such as Candida albicans is of critical importance, yet progress can be hindered by challenges associated with manipulating these pathogens genetically. CRISPR-based technologies have significantly improved our ability to manipulate the genomes of countless organisms, including fungal pathogens such as C. albicans. CRISPR interference (CRISPRi) is a modified variation of CRISPR technology that enables the targeted genetic repression of specific genes of interest and can be used as a technique for studying essential genes. We recently developed tools to enable CRISPRi in C. albicans and the repression of essential genes in this fungus. Here, we describe a protocol for CRISPRi in C. albicans, including the design of the single-guide RNAs (sgRNAs) for targeting essential genes, the high-efficiency cloning of sgRNAs into C. albicans-optimized CRISPRi plasmids, transformation into fungal strains, and testing to monitor the repression capabilities of these constructs. Together, this protocol will illuminate efficient strategies for targeted genetic repression of essential genes in C. albicans using a novel CRISPRi platform.