Abstract
ABSTRACTCandida 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.
Highlights
Candida auris is a multidrug-resistant human fungal pathogen that has recently emerged worldwide
The double-strand break (DSB) can be repaired by errorprone nonhomologous end joining (NHEJ) or by homology-directed repair (HDR), the latter relying on user-supplied repair templates to make mutations as small as a single base pair in size or to incorporate large heterologous sequences
This system utilizes Cas9 and guide RNA (gRNA) expression cassettes that are either amplified or digested from plasmids and integrated within the LEU2 locus to create a LEU2 disruption strain that is resistant to nourseothricin or hygromycin B and auxotrophic for leucine (Fig. 1A)
Summary
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%. 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. IMPORTANCE Candida auris is a recently emerged multidrug-resistant fungal pathogen capable of causing life-threatening systemic infections in humans. Since its first application in Candida albicans in 2015 [17], CRISPR/Cas9-mediated genome editing has greatly increased the speed and efficiency of creating mutations in several Candida species (reviewed in reference 18) These systems rely on a unique RNA-guided DNA nuclease that directs a double-strand break (DSB) at a locus of interest. The DSB can be repaired by errorprone nonhomologous end joining (NHEJ) or by homology-directed repair (HDR), the latter relying on user-supplied repair templates to make mutations as small as a single base pair in size or to incorporate large heterologous sequences
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