INTRODUCTION
Non-albicans -Candida (NAC) species are a rising concern for immunocompromised patients, and Candida glabrata is the major NAC species in many developed countries (Rodrigues et al. , 2014). The Candida genus is not monophyletic, and C. glabrata is not part of the major clade that includes C. albicans (Dujon et al ., 2004). Instead, it is part of theNakaseomyces clade, which includes two other human pathogenic species in addition to C. glabrata ; Candida bracarensis and Candida nivariensis (Gabaldon et al ., 2013). TheNakaseomyces clade is quite closely related to the model yeastSaccharomyces cerevisiae (Dujon et al. , 2004), and thus many molecular tools such as marker genes or replication origins, can be transferred from S. cerevisiae to C. glabrata (Muller et al. , 2007).
The CRISPR-Cas9 system (Knott and Doudna, 2018) has been adapted to many species, including yeasts, to induce chromosome Double-Strand Breaks (DSBs) at loci targeted through the guide RNA (gRNA), most often with the aim of editing genomic sequences. CRISPR-Cas9 has indeed been adapted to C . glabrata (Cen et al. , 2017; Enkleret al. , 2016; Maroc et al, 2019; Vyas et al. , 2018), creating DSBs that can be repaired by either Non-Homologous End-Joining (NHEJ) or Homologous Recombination (HR) when a template homologous to the cut-site is provided. Using CRISPR-Cas9 for genome editing generally relies on constitutive promoters for the Cas9 gene and the gRNA, but the use of inducible expression allows the study of repair of DSBs, for example in the model yeast S. cerevisiae (Lemos et al. , 2018). Inducible expression is also particularly useful in species such as the Nakaseomyces Candida. Because these are asexual and haploid, the fact that a gene is essential can never be proven by tetrad analysis after sporulation of a heterozygous diploid deletant. It is also difficult, when deletion experiments yield no transformants, to determine whether this is because the gene is essential or because experiments fail for technical reasons. On the other hand, when the creation of a DSB for gene editing is controlled in living cells, a negative result, ie when no mutants are obtained in the targeted gene, can be interpreted with more confidence as proving the essential nature of the gene.
We have developed an inducible system CRISPR-Cas9 (Maroc and Fairhead, 2019) based on the MET3 promoter, and with the URA3 gene as marker, both sequences coming from C. glabrata (Zordanet al. , 2013). We have previously shown that this can induce DSBs in this species, by targeting the ADE2 gene. This gene is involved in the de novo purine nucleotide synthesis pathway, and its inactivation causes the accumulation of a red pigment in cells, leading to a red colony color in S. cerevisiae (Dorfman, 1969) and the Candida tested here (Maroc and Fairhead, 2019; and this work). We have since constructed a new version of the expression plasmid, because the original one contained sequence repeats that led to frequent rearrangements when cloned in E. coli (see Materials and Methods).
We report here the use of this new inducible CRISPR-Cas9 system in the pathogens C. glabrata, C. bracarensis, and C. nivariensis . Cas9 creates a DSB at the ADE2 target locus, that is repaired by NHEJ. We confirm that expression of a CRISPR-Cas9 targeting ADE2 does not affect viability on plates in the C. glabrata , although we observe a decrease in viability in the first hours of induction in liquid medium. For the other two Candida species, survival is lower. We also report that repair of the DSB by unfaithful NHEJ results in different sequence modifications in C. glabrata as compared to the two other species.