A single Ho-induced double-strand break at the MAT locus is lethal in Candida glabrata.

Mating-type switching is a complex mechanism that promotes sexual reproduction in Saccharomycotina. In the model species Saccharomyces cerevisiae, mating-type switching is initiated by the Ho endonuclease that performs a site-specific double-strand break (DSB) at MAT, repaired by homologous recombination (HR) using one of the two silent mating-type loci, HMLalpha and HMRa. The reasons why all the elements of the mating-type switching system have been conserved in some Saccharomycotina, that do not show a sexual cycle nor mating-type switching, remain unknown. To gain insight on this phenomenon, we used the yeast Candida glabrata, phylogenetically close to S. cerevisiae, and for which no spontaneous and efficient mating-type switching has been observed. We have previously shown that expression of S. cerevisiae's Ho (ScHo) gene triggers mating-type switching in C. glabrata, but this leads to massive cell death. In addition, we unexpectedly found, that not only MAT but also HML was cut in this species, suggesting the formation of multiple chromosomal DSBs upon HO induction. We now report that HMR is also cut by ScHo in wild-type strains of C. glabrata. To understand the link between mating-type switching and cell death in C. glabrata, we constructed strains mutated precisely at the Ho recognition sites. We find that even when HML and HMR are protected from the Ho-cut, introducing a DSB at MAT is sufficient to induce cell death, whereas one DSB at HML or HMR is not. We demonstrate that mating-type switching in C. glabrata can be triggered using CRISPR-Cas9, without high lethality. We also show that switching is Rad51-dependent, as in S. cerevisiae, but that donor preference is not conserved in C. glabrata. Altogether, these results suggest that a DSB at MAT can be repaired by HR in C. glabrata, but that repair is prevented by ScHo.

Lessons from the Nakaseomyces: mating-type switching, DSB repair and evolution of Ho.

This short paper aims to review what our recent studies in the Nakaseomyces yeasts, principally Candida glabrata, reveal about the evolution of the mating-type switching system and its components, as well as about the repair of chromosomal double-strand breaks in this clade. In the model yeast Saccharomyces cerevisiae, the study of mating-type switching has, over the years, led to major discoveries in how cells process chromosomal breaks. Indeed, in this species, switching, which allows every haploid cell to produce cells of opposite mating types that can mate together, is initiated by the Ho endonuclease, linking sexual reproduction to a programmed chromosomal cut. More recently, the availability of other yeasts' genomes from type strains and from populations, and the ability to manipulate and edit the genomes of most yeasts in the laboratory, has enabled scientists to explore mating-type switching in new species, thus enriching our evolutionary perspective on this phenomenon. In this review, we will show how the study of mating-type switching in C. glabrata and Nakaseomyces delphensis has allowed us to reveal possible additional roles for Ho, and also to discover major differences in DSB repair at central and subtelomeric sexual loci. In addition, we report how the study of repair of chromosomal breaks induced by CRISPR-Cas9 reveals that efficient and faithful NHEJ is a major repair pathway in C. glabrata.

A new inducible CRISPR-Cas9 system useful for genome editing and study of double-strand break repair in Candida glabrata.

In recent years, the CRISPR-Cas9 system has proven extremely useful for genome editing in many species, including the model yeast Saccharomyces cerevisiae and other yeast species such as Candida glabrata. Inducible CRISPR-Cas9 systems have the additional advantage of allowing to separate the transformation step of the organism by the CRISPR-Cas9 system, from the cutting and repair steps. This has indeed been developed in S. cerevisiae, where most inducible expression systems rely on the GAL promoters. Unfortunately, C. glabrata is gal- and lacks the GAL genes, like many other yeast species. We report here the use of a vector expressing cas9 under the control of the MET3 promoter, with the guide RNA cloned into the same plasmid. We show that it can be used efficiently in C. glabrata, for both described outcomes of CRISPR-Cas9-induced chromosome breaks; nonhomologous end joining in the absence of a homologous repair template; and homologous recombination in the presence of such a template. This system therefore allows easy editing of the genome of C. glabrata, and its inducibility may allow identification of essential genes in this asexual yeast, where spore lethality cannot be observed, as well as the study of double-strand break repair.

Functional genetic characterization of stress tolerance and biofilm formation in Nakaseomyces (Candida) glabrata via a novel CRISPR activation system.

The overexpression of genes frequently arises in Nakaseomyces (formerly Candida) glabrata via gain-of-function mutations, gene duplication, or aneuploidies, with important consequences on pathogenesis traits and antifungal drug resistance. This highlights the need to develop specific genetic tools to mimic and study genetic amplification in this important fungal pathogen. Here, we report the development, validation, and applications of the first clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system in N. glabrata for targeted genetic overexpression. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in N. glabrata, and further assess optimal guide RNA targeting for robust overexpression. We demonstrate the applications of CRISPRa to overexpress genes involved in fungal pathogenesis and drug resistance and detect corresponding phenotypic alterations in these key traits, including the characterization of novel phenotypes. Finally, we capture strain variation using our CRISPRa system in two commonly used N. glabrata genetic backgrounds. Together, this tool will expand our capacity for functional genetic overexpression in this pathogen, with numerous possibilities for future applications.IMPORTANCENakaseomyces (formerly Candida) glabrata is an important fungal pathogen that is now the second leading cause of candidiasis infections. A common strategy that this pathogen employs to resist antifungal treatment is through the upregulation of gene expression, but we have limited tools available to study this phenomenon. Here, we develop, optimize, and apply the use of CRISPRa as a means to overexpress genes in N. glabrata. We demonstrate the utility of this system to overexpress key genes involved in antifungal susceptibility, stress tolerance, and biofilm growth. This tool will be an important contribution to our ability to study the biology of this important fungal pathogen.

High-content CRISPR screening.

CRISPR screens are a powerful source of biological discovery, enabling the unbiased interrogation of gene function in a wide range of applications and species. In pooled CRISPR screens, various genetically encoded perturbations are introduced into pools of cells. The targeted cells proliferate under a biological challenge such as cell competition, drug treatment or viral infection. Subsequently, the perturbation-induced effects are evaluated by sequencing-based counting of the guide RNAs that specify each perturbation. The typical results of such screens are ranked lists of genes that confer sensitivity or resistance to the biological challenge of interest. Contributing to the broad utility of CRISPR screens, adaptations of the core CRISPR technology make it possible to activate, silence or otherwise manipulate the target genes. Moreover, high-content read-outs such as single-cell RNA sequencing and spatial imaging help characterize screened cells with unprecedented detail. Dedicated software tools facilitate bioinformatic analysis and enhance reproducibility. CRISPR screening has unravelled various molecular mechanisms in basic biology, medical genetics, cancer research, immunology, infectious diseases, microbiology and other fields. This Primer describes the basic and advanced concepts of CRISPR screening and its application as a flexible and reliable method for biological discovery, biomedical research and drug development - with a special emphasis on high-content methods that make it possible to obtain detailed biological insights directly as part of the screen.