Faculty Opinions recommendation of A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families.

Candida albicansis a pathogenic yeast that causes mucosal and systematic infections with high mortality. The absence of facile molecular genetics has been a major impediment to analysis of pathogenesis. The lack of meiosis coupled with the absence of plasmids makes genetic engineering cumbersome, especially for essential functions and gene families. We describe aC. albicansCRISPR system that overcomes many of the obstacles to genetic engineering in this organism. The high frequency with which CRISPR-induced mutations can be directed to target genes enables easy isolation of homozygous gene knockouts, even without selection. Moreover, the system permits the creation of strains with mutations in multiple genes, gene families, and genes that encode essential functions. This CRISPR system is also effective in a fresh clinical isolate of undetermined ploidy. Our method transforms the ability to manipulate the genome ofCandidaand provides a new window into the biology of this pathogen.

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Different expression levels of ALS and SAP genes contribute to recurrent vulvovaginal candidiasis by Candida albicans

Future Microbiology ◽

10.2217/fmb-2020-0059 ◽

2021 ◽

Author(s):

Patrícia de S Bonfim-Mendonça ◽

Flávia K Tobaldini-Valério ◽

Isis RG Capoci ◽

Daniella R Faria ◽

Karina M Sakita ◽

...

Keyword(s):

Candida Albicans ◽

Hela Cells ◽

Gene Families ◽

Vulvovaginal Candidiasis ◽

Aspartic Proteinases ◽

Expression Levels ◽

Host Interactions ◽

Qrt Pcr ◽

Colony Forming Units ◽

Number Of Genes

Aim: To study the behavior of Candida albicans from women with vulvovaginal candidiasis (VVC), recurrent VVC (RVVC) and asymptomatic (AS), regarding adhesion on HeLa cells and their ability to express secreted aspartic proteinases ( SAP) genes, agglutinin like sequence ( ALS) genes and HWP1. Materials & methods: The adhesion of Candida albicans to HeLa cells was evaluated by colony-forming units, and the expressed genes were evaluated by qRT-PCR. Results: AS and VVC isolates showed greater ability to adhere HeLa cells when compared with RVVC isolate. Nevertheless, RVVC isolate exhibited upregulation of a large number of genes of ALS and SAP gene families and HWP1 gene. Conclusion: The results demonstrated that RVVC isolate expressed significantly important genes for invasion and yeast–host interactions.

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A novel CRISPRi platform to study the role of essential genes in antifungal drug-resistant Candida albicans isolates

Access Microbiology ◽

10.1099/acmi.cc2021.po0157 ◽

2021 ◽

Vol 3 (12) ◽

Author(s):

Lauren Wensing ◽

Rebecca Shapiro ◽

Deeva Uthayakumar ◽

Viola Halder ◽

Jehoshua Sharma ◽

...

Keyword(s):

Candida Albicans ◽

Transcriptional Repression ◽

Drug Targets ◽

Large Scale ◽

Essential Gene ◽

Essential Genes ◽

Antifungal Drugs ◽

Antifungal Drug ◽

Drug Resistant ◽

Fungal Organisms

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.

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Generation of conditional lethal Candida albicans mutants by inducible deletion of essential genes

Molecular Microbiology ◽

10.1046/j.1365-2958.2002.03167.x ◽

2002 ◽

Vol 46 (1) ◽

pp. 269-280 ◽

Cited By ~ 37

Author(s):

Sonja Michel ◽

Sophia Ushinsky ◽

Bert Klebl ◽

Ekkehard Leberer ◽

David Thomas ◽

...

Keyword(s):

Candida Albicans ◽

Essential Genes

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Independent regulation of chitin synthase and chitinase activity in Candida albicans and Saccharomyces cerevisiae

Microbiology ◽

10.1099/mic.0.26661-0 ◽

2004 ◽

Vol 150 (4) ◽

pp. 921-928 ◽

Cited By ~ 55

Author(s):

Serena Selvaggini ◽

Carol A. Munro ◽

Serge Paschoud ◽

Dominique Sanglard ◽

Neil A. R. Gow

Keyword(s):

Saccharomyces Cerevisiae ◽

Candida Albicans ◽

De Novo ◽

Gene Families ◽

Chitinase Activity ◽

Chitin Synthase ◽

Chitin Synthesis ◽

Growing Cell ◽

Chitinase Genes

Chitin is an essential structural polysaccharide in fungi that is required for cell shape and morphogenesis. One model for wall synthesis at the growing cell surface suggests that the compliance that is necessary for turgor-driven expansion of the cell wall involves a delicate balance of wall synthesis and lysis. Accordingly, de novo chitin synthesis may involve coordinated regulation of members of the CHS chitin synthase and CHT chitinase gene families. To test this hypothesis, the chitin synthase and chitinase activities of cell-free extracts were measured, as well as the chitin content of cell walls isolated from isogenic mutant strains that contained single or multiple knock-outs in members of these two gene families, in both Candida albicans and Saccharomyces cerevisiae. However, deletion of chitinase genes did not markedly affect specific chitin synthase activity, and deletion of single CHS genes had little effect on in vitro specific chitinase activity in either fungus. Chitin synthesis and chitinase production was, however, regulated in C. albicans during yeast–hypha morphogenesis. In C. albicans, the total specific activities of both chitin synthase and chitinase were higher in the hyphal form, which was attributable mainly to the activities of Chs2 and Cht3, respectively. It appeared, therefore, that chitin synthesis and hydrolysis were not coupled, but that both were regulated during yeast–hypha morphogenesis in C. albicans.

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Systematic Analysis of Functionally Related Gene Clusters in the Opportunistic Pathogen, Candida albicans

Microorganisms ◽

10.3390/microorganisms9020276 ◽

2021 ◽

Vol 9 (2) ◽

pp. 276

Author(s):

Sarah Asfare ◽

Reem Eldabagh ◽

Khizar Siddiqui ◽

Bharvi Patel ◽

Diellza Kaba ◽

...

Keyword(s):

Gene Expression ◽

Candida Albicans ◽

Gene Families ◽

Gene Clusters ◽

Opportunistic Pathogen ◽

Systematic Analysis ◽

Genomic Arrangement ◽

A Genome ◽

Opportunistic Human Pathogen ◽

Organismal Development

The proper balance of gene expression is essential for cellular health, organismal development, and maintaining homeostasis. In response to complex internal and external signals, the cell needs to modulate gene expression to maintain proteostasis and establish cellular identity within its niche. On a genome level, single-celled prokaryotic microbes display clustering of co-expressed genes that are regulated as a polycistronic RNA. This phenomenon is largely absent from eukaryotic microbes, although there is extensive clustering of co-expressed genes as functional pairs spread throughout the genome in Saccharomyces cerevisiae. While initial analysis demonstrated conservation of clustering in divergent fungal lineages, a comprehensive analysis has yet to be performed. Here we report on the prevalence, conservation, and significance of the functional clustering of co-regulated genes within the opportunistic human pathogen, Candida albicans. Our analysis reveals that there is extensive clustering within this organism—although the identity of the gene pairs is unique compared with those found in S. cerevisiae—indicating that this genomic arrangement evolved after these microbes diverged evolutionarily, rather than being the result of an ancestral arrangement. We report a clustered arrangement in gene families that participate in diverse molecular functions and are not the result of a divergent orientation with a shared promoter. This arrangement coordinates the transcription of the clustered genes to their neighboring genes, with the clusters congregating to genomic loci that are conducive to transcriptional regulation at a distance.

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Gene Essentiality Analyzed byIn VivoTransposon Mutagenesis and Machine Learning in a Stable Haploid Isolate ofCandida albicans

mBio ◽

10.1128/mbio.02048-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 42

Author(s):

Ella Shtifman Segal ◽

Vladimir Gritsenko ◽

Anton Levitan ◽

Bhawna Yadav ◽

Naama Dror ◽

...

Keyword(s):

Machine Learning ◽

Candida Albicans ◽

Fungal Pathogens ◽

Essential Genes ◽

Antifungal Drugs ◽

Model Organisms ◽

Standard Laboratory ◽

Genome Database ◽

Content Type ◽

Laboratory Conditions

ABSTRACTKnowing the full set of essential genes for a given organism provides important information about ways to promote, and to limit, its growth and survival. For many non-model organisms, the lack of a stable haploid state and low transformation efficiencies impede the use of conventional approaches to generate a genome-wide comprehensive set of mutant strains and the identification of the genes essential for growth. Here we report on the isolation and utilization of a highly stable haploid derivative of the human pathogenic fungusCandida albicans, together with a modified heterologous transposon and machine learning (ML) analysis method, to predict the degree to which all of the open reading frames are required for growth under standard laboratory conditions. We identified 1,610 C. albicansessential genes, including 1,195 with high “essentiality confidence” scores, thereby increasing the number of essential genes (currently 66 in the Candida Genome Database) by >20-fold and providing an unbiased approach to determine the degree of confidence in the determination of essentiality. Among the genes essential inC. albicanswere 602 genes also essential in the model budding and fission yeasts analyzed by both deletion and transposon mutagenesis. We also identified essential genes conserved among the four major human pathogensC. albicans,Aspergillus fumigatus,Cryptococcus neoformans, andHistoplasma capsulatumand highlight those that lack homologs in humans and that thus could serve as potential targets for the design of antifungal therapies.IMPORTANCEComprehensive understanding of an organism requires that we understand the contributions of most, if not all, of its genes. Classical genetic approaches to this issue have involved systematic deletion of each gene in the genome, with comprehensive sets of mutants available only for very-well-studied model organisms. We took a different approach, harnessing the power ofin vivotransposition coupled with deep sequencing to identify >500,000 different mutations, one per cell, in the prevalent human fungal pathogenCandida albicansand to map their positions across the genome. The transposition approach is efficient and less labor-intensive than classic approaches. Here, we describe the production and analysis (aided by machine learning) of a large collection of mutants and the comprehensive identification of 1,610 C. albicansgenes that are essential for growth under standard laboratory conditions. Among theseC. albicansessential genes, we identify those that are also essential in two distantly related model yeasts as well as those that are conserved in all four major human fungal pathogens and that are not conserved in the human genome. This list of genes with functions important for the survival of the pathogen provides a good starting point for the development of new antifungal drugs, which are greatly needed because of the emergence of fungal pathogens with elevated resistance and/or tolerance of the currently limited set of available antifungal drugs.

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Genome Editing of Babesia bovis Using the CRISPR/Cas9 System

mSphere ◽

10.1128/msphere.00109-19 ◽

2019 ◽

Vol 4 (3) ◽

Cited By ~ 3

Author(s):

Hassan Hakimi ◽

Takahiro Ishizaki ◽

Yuto Kegawa ◽

Osamu Kaneko ◽

Shin-ichiro Kawazu ◽

...

Keyword(s):

Functional Analysis ◽

Genetic Engineering ◽

Genome Editing ◽

Gene Families ◽

Babesia Bovis ◽

Whole Genome Sequence ◽

Meat Production ◽

Bovine Babesiosis ◽

Body Protein ◽

Content Type

ABSTRACT Babesia bovis, the most virulent causative agent of bovine babesiosis, is prevalent in tropical and subtropical regions of the world. Although the whole-genome sequence was released more than a decade ago, functional analysis of the genomics of this parasite is hampered by the limited breadth of genetic engineering tools. In this study, we implemented the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system for B. bovis and demonstrated its potential for genome editing. Cas9 and human dihydrofolate reductase (hDHFR) were simultaneously expressed by the B. bovis elongation factor-1α bidirectional promoter, and a single guide RNA was expressed via the B. bovis U6 spliceosomal RNA promoter. Using a single plasmid construct, we were able to add an epitope tag to spherical body protein 3 (SBP3), introduce a point mutation into thioredoxin peroxidase 1 (tpx-1) to impair the function of the product, and replace the tpx-1 open reading frame with the other protein. Epitope tagging of SBP3 was efficient using this system, with a negligible number of remaining wild-type parasites and a pure transgenic population produced by allelic replacement of tpx-1. This advancement in genetic engineering tools for B. bovis will aid functional analysis of the genome and underpin characterization of candidate drug and vaccine targets. IMPORTANCE Babesia bovis is the most virulent cause of bovine babesiosis worldwide. The disease consequences are death, abortion, and economical loss due to reduced milk and meat production. Available vaccines are not effective, treatment options are limited, and emergence of drug and acaricide resistance has been reported from different regions. There is an urgent need to identify new drug and vaccine targets. Greater than half of the genes in B. bovis genome, including several expanded gene families which are unique for Babesia spp., have no predicted function. The available genetic engineering tools are based on conventional homologous recombination, which is time-consuming and inefficient. In this study, we adapted the CRISPR/Cas9 system as a robust genetic engineering tool for B. bovis. This advancement will aid future functional studies of uncharacterized genes.

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