xpanding 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. IMPORTANCEThe 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. We have demonstrated the effectiveness of an expression-free CRISPR-Cas9 RNP-based approach employing 130-150 bp homology regions for directed repair. Our approach is robust and efficient for making gene deletions in C. glabrata, C. auris and C. albicans as well as epitope tagging in C. glabrata. Furthermore, we demonstrated that KanMX and BleMX drug resistance cassettes can be repurposed in C. glabrata and BleMX in C. auris. Overall, we have expanded the toolkit for genetic manipulation and discovery in fungal pathogens.