A Markerless CRISPR-Mediated System for Genome Editing in Candida auris Reveals a Conserved Role for Cas5 in the Caspofungin Response

Abstract

Candida auris is a recently emerged multidrug-resistant fungal pathogen capable of causing life-threatening systemic infections in humans. Few tools are available for genome editing in C. auris . Candida auris is a multidrug-resistant human fungal pathogen that has recently emerged worldwide. It can cause life-threatening disseminated infections in humans, with mortality rates upwards of 50%. The molecular mechanisms underlying its multidrug resistance and pathogenic properties are largely unknown. Few methods exist for genome editing in C. auris , all of which rely on selectable markers that limit the number of modifications that can be made. Here, we present a markerless CRISPR/Cas9-mediated genome editing system in C. auris . Using this system, we successfully deleted genes of interest and subsequently reconstituted them at their native loci in isolates across all five C. auris clades. This system also enabled us to introduce precision genome edits to create translational fusions and single point mutations. Using Cas5 as a test case for this system, we discovered a conserved role for Cas5 in the caspofungin response between Candida albicans and C. auris . Overall, the development of a system for precise and facile genome editing in C. auris that can allow edits to be made in a high-throughput manner is a major step forward in improving our understanding of this important human fungal pathogen. IMPORTANCE Candida auris is a recently emerged multidrug-resistant fungal pathogen capable of causing life-threatening systemic infections in humans. Few tools are available for genome editing in C. auris . Here, we present a markerless genome editing system for C. auris that relies on CRISPR/Cas9 technology and works to modify the genomes of all known C. auris clades. Using this system, we discovered a conserved role for Cas5 in the caspofungin response between C. albicans and C. auris . Overall, the development of a system for facile genome editing in C. auris is a major step forward in improving our understanding of this important human fungal pathogen.