CRISPR-Cas9 RNP-Mediated Deletion of ERG25 in Non-albicans Candida Species, Including Candida auris.

The incidence of infections caused by non-albicans Candida (NAC) species, including Candida glabrata and Candida tropicalis, has recently increased. Furthermore, Candida auris, a multidrug-resistant yeast, poses a serious threat to global health. The development of novel antifungal agents with alternative mechanisms of action is necessary to combat these fungi. However, genetic analyses of the virulence factors in these NAC species are insufficient for this purpose. Recent advancements in the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system have facilitated enhanced the genetic analysis of NAC species. The RNP-based system, in which the Cas9-gRNA complex is assembled in vitro and introduced into cells, offers a simplified approach to genetic modification, eliminating the need for species-specific plasmids. Previous our research identified the ERG25 gene, which encodes C-4 sterol methyl oxidase, as a promising antifungal target in C. glabrata. This study demonstrated deletion of the ERG25 homolog in C. glabrata and C. auris using an RNP-based CRISPR-Cas9 system. The deletion of ERG25 in C. auris and C. glabrata indicated that Erg25 is crucial for the survival of these pathogenic yeasts within the host. Furthermore, we have successfully deleted the ERG25 alleles in C. tropicalis and Candida parapsilosis, demonstrating the effectiveness of using both the CRISPR-Cas9 and Cre-loxP systems in these species for the first time.