Fungal pathogens are an increasingly important cause of human disease and mortality, and Candida albicans is among the most common causes of fungal disease. Studying this important fungal pathogen requires a comprehensive genetic toolkit to establish how different genetic factors play roles in the biology and virulence of this pathogen. Here, we developed a CRISPR-based genetic regulation platform to achieve targeted repression of C. albicans genes. This CRISPR interference (CRISPRi) technology exploits a nuclease-dead Cas9 protein (dCas9) fused to transcriptional repressors. The dCas9 fusion proteins pair with a guide RNA to target genetic promoter regions and to repress expression from these genes. We demonstrated the functionality of this system for repression in C. albicans and show that we can apply this technology to repress essential genes. Taking the results together, this work presents a new technology for efficient genetic repression in C. albicans, with important applications for genetic analysis in this fungal pathogen.

Fungal infections are a growing public health concern, and an increasingly important cause of human mortality, with Candida species being amongst the most frequently encountered of these opportunistic fungal pathogens. Several Candida species are polymorphic, and able to transition between distinct morphological states, including yeast, hyphal, and pseudohyphal forms. While not all Candida pathogens are polymorphic, the ability to undergo morphogenesis is linked with the virulence of many of these pathogens. There are also many connections between Candida morphogenesis and antifungal drug treatment and susceptibility. Here, we review how Candida morphogenesis—a key virulence trait—is linked with antifungal drugs and antifungal drug resistance. We highlight how antifungal therapeutics are able to modulate morphogenesis in both sensitive and drug-resistant Candida strains, the shared signaling pathways that mediate both morphogenesis and the cellular response to antifungal drugs and drug resistance, and the connection between Candida morphology, drug resistance, and biofilm growth. We further review the development of anti-virulence drugs, and targeting Candida morphogenesis as a novel therapeutic strategy to target fungal pathogens. Together, this review highlights important connections between fungal morphogenesis, virulence, and susceptibility to antifungals.

The Candida genus encompasses a diverse group of ascomycete fungi that have captured the attention of the scientific community, due to both their role in pathogenesis and emerging applications in biotechnology; the development of gene editing tools such as CRISPR, to analyze fungal genetics and perform functional genomic studies in these organisms, is essential to fully understand and exploit this genus, to further advance antifungal drug discovery and industrial value. However, genetic manipulation of Candida species has been met with several distinctive barriers to progress, such as unconventional codon usage in some species, as well as the absence of a complete sexual cycle in its diploid members. Despite these challenges, the last few decades have witnessed an expansion of the Candida genetic toolbox, allowing for diverse genome editing applications that range from introducing a single point mutation to generating large-scale mutant libraries for functional genomic studies. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology is among the most recent of these advancements, bringing unparalleled versatility and precision to genetic manipulation of Candida species. Since its initial applications in Candida albicans, CRISPR-Cas9 platforms are rapidly evolving to permit efficient gene editing in other members of the genus. The technology has proven useful in elucidating the pathogenesis and host-pathogen interactions of medically relevant Candida species, and has led to novel insights on antifungal drug susceptibility and resistance, as well as innovative treatment strategies. CRISPR-Cas9 tools have also been exploited to uncover potential applications of Candida species in industrial contexts. This review is intended to provide a historical overview of genetic approaches used to study the Candida genus and to discuss the state of the art of CRISPR-based genetic manipulation of Candida species, highlighting its contributions to deciphering the biology of this genus, as well as providing perspectives for the future of Candida genetics.

For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPRa to overexpress genes involved in pathogenesis and drug susceptibility and contribute towards the identification of novel phenotypes. Consequently, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.

Fungi are nature’s recyclers, allowing for ecological nutrient cycling and, in turn, the continuation of life on Earth. Some fungi inhabit the human microbiome where they can provide health benefits, while others are opportunistic pathogens that can cause disease. Yeasts, members of the fungal kingdom, have been domesticated by humans for the production of beer, bread, and, recently, medicine and chemicals. Still, the great untapped potential exists within the diverse fungal kingdom. However, many yeasts are intractable, preventing their use in biotechnology or in the development of novel treatments for pathogenic fungi. Therefore, as a first step for the domestication of new fungi, an efficient DNA delivery method needs to be developed. Here, we report the creation of superior conjugative plasmids and demonstrate their transfer via conjugation from bacteria to 7 diverse yeast species including the emerging pathogen Candida auris. To create our superior plasmids, derivatives of the 57 kb conjugative plasmid pTA-Mob 2.0 were built using designed gene deletions and insertions, as well as some unintentional mutations. Specifically, a cluster mutation in the promoter of the conjugative gene traJ had the most significant effect on improving conjugation to yeasts. In addition, we created Golden Gate assembly-compatible plasmid derivatives that allow for the generation of custom plasmids to enable the rapid insertion of designer genetic cassettes. Finally, we demonstrated that designer conjugative plasmids harboring engineered restriction endonucleases can be used as a novel antifungal agent, with important applications for the development of next-generation antifungal therapeutics.

For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness genetic tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans. Our CRISPRa system exploits a nuclease-dead dCas9 fused to the tripartite activator complex VP64-p65-Rta (VPR), and a Golden Gate site for efficient guide RNA cloning to overexpress genes of interest. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust overexpression. We further demonstrate the application of CRISPRa to overexpress genes involved in fungal pathogenesis and drug resistance and detect corresponding phenotypic alterations in these traits. Together, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.

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With the emergence of antifungal resistant Candida albicans strains, the need for new antifungal drugs is critical in combating this fungal pathogen. Investigating essential genes in C. albicans is a vital step in characterizing putative antifungal drug targets. As some of these essential genes are conserved between fungal organisms, developed therapies targeting these genes have the potential to be broad range antifungals. In order to study these essential genes, classical genetic knockout or CRISPR-based approaches cannot be used as disrupting essential genes leads to lethality in the organism. Fortunately, a variation of the CRISPR system (CRISPR interference or CRISPRi) exists that enables precise transcriptional repression of the gene of interest without introducing genetic mutations. CRISPRi utilizes an endonuclease dead Cas9 protein that can be targeted to a precise location but lacks the ability to create a double-stranded break. The binding of the dCas9 protein to DNA prevents the binding of RNA polymerase to the promoter through steric hindrance thereby reducing expression. We recently published the novel use of this technology in C. albicans and are currently working on expanding this technology to large scale repression of essential genes. Through the construction of an essential gene CRISPRi-sgRNA library, we can begin to study the function of essential genes under different conditions and identify genes that are involved in critical processes such as drug tolerance in antifungal resistant background strains. These genes can ultimately be characterized as putative targets for novel antifungal drug development, or targeted as a means to sensitize drug-resistant strains to antifungal treatment.