Expanding the toolkit for genetic manipulation and discovery inCandidaspecies using a CRISPR ribonucleoprotein-based approach

Abstract

ABSTRACTThe World Health Organization recently published the first list of priority fungal pathogens highlighting multipleCandidaspecies includingC. glabrata,C. albicans, andC. auris. The use of CRISPR-Cas9 and auxotrophicC. glabrataandC. albicansstrains 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,NatMXandHphMX. Using anin vitroassembled CRISPR-Cas9 ribonucleoprotein (RNP)-based system and 130-150 bp homology regions for directed repair, we expand the drug resistance cassettes forCandidato includeKanMXandBleMX, commonly used inS. cerevisiae. As a proof of principle, we demonstrated efficient deletion ofERGgenes usingKanMXandBleMX. 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 ofERGgenes using an existingKanMXcassette. This indicates that CRISPR-Cas9 RNP can be used to repurpose theS. cerevisiaetoolkit. Furthermore, we demonstrated that this method is effective at deletingERG3inC. aurisusing a codon optimizedBleMXcassette and effective at deleting the epigenetic factor,SET1, inC. albicansusing a recyclableSAT1.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 inC. glabrata,C. aurisandC. albicansas well as epitope tagging inC. glabrata. Furthermore, we demonstrated thatKanMXandBleMXdrug resistance cassettes can be repurposed inC. glabrataandBleMXinC. auris. Overall, we have expanded the toolkit for genetic manipulation and discovery in fungal pathogens.