Expanding the toolkit for genetic manipulation and discovery in Candida species using a CRISPR ribonucleoprotein-based approach.

The World Health Organization recently published the first list of priority fungal pathogens highlighting multiple Candida species including C. glabrata, C. albicans, and C. auris. The use of CRISPR-Cas9 and auxotrophic C. glabrata and C. albicans strains have been instrumental in the study of these fungal pathogens. Dominant drug resistance cassettes are also critical for genetic manipulation and eliminate the concern of altered virulence when using auxotrophic strains. However, genetic manipulation has been mainly limited to the use of two drug resistance cassettes, NatMX and HphMX. Using an in vitro assembled CRISPR-Cas9 ribonucleoprotein (RNP)-based system and 130-150 bp homology regions for directed repair, we expand the drug resistance cassettes for Candida to include KanMX and BleMX, commonly used in S. cerevisiae. As a proof of principle, we demonstrated efficient deletion of ERG genes using KanMX and BleMX. We also showed the utility of the CRISPR-Cas9 RNP system for generating double deletions of genes in the ergosterol pathway and endogenous epitope tagging of ERG genes using an existing KanMX cassette. This indicates that CRISPR-Cas9 RNP can be used to repurpose the S. cerevisiae toolkit. Furthermore, we demonstrated that this method is effective at deleting ERG3 in C. auris using a codon optimized BleMX cassette and effective at deleting the epigenetic factor, SET1, in C. albicans using a recyclable SAT1. Using this expanded toolkit, we discovered new insights into fungal biology and drug resistance.

An expanded toolkit of drug resistance cassettes for Candida glabrata, Candida auris, and Candida albicans leads to new insights into the ergosterol pathway.

IMPORTANCE: The increasing problem of drug resistance and emerging pathogens is an urgent global health problem that necessitates the development and expansion of tools for studying fungal drug resistance and pathogenesis. Prior studies in Candida glabrata, Candida auris, and Candida albicans have been mainly limited to the use of NatMX/SAT1 and HphMX/CaHyg for genetic manipulation in prototrophic strains and clinical isolates. In this study, we demonstrated that NatMX/SAT1, HphMX, KanMX, and/or BleMX drug resistance cassettes when coupled with a CRISPR-ribonucleoprotein (RNP)-based system can be efficiently utilized for deleting or modifying genes in the ergosterol pathway of C. glabrata, C. auris, and C. albicans. Moreover, the utility of these tools has provided new insights into ERG genes and their relationship to azole resistance in Candida. Overall, we have expanded the toolkit for Candida pathogens to increase the versatility of genetically modifying complex pathways involved in drug resistance and pathogenesis.