SpRY Cas9 Can Utilize a Variety of Protospacer Adjacent Motif Site Sequences To Edit the Candida albicans Genome

ABSTRACT Candida albicans is a human fungal pathogen capable of causing life-threatening infections. The ability to edit the C. albicans genome using CRISPR/Cas9 is an important tool investigators can leverage in their search for novel therapeutic targets. However, wild-type Cas9 requires an NGG protospacer adjacent motif (PAM), leaving many AT-rich regions of DNA inaccessible. A recently described near-PAMless CRISPR system that utilizes the SpRY Cas9 variant can target non-NGG PAM sequences. Using this system as a model, we developed C. albicans CRISPR/SpRY. We tested our system by mutating C. albicans ADE2 and show that CRISPR/SpRY can utilize non-NGG PAM sequences in C. albicans. Our CRISPR/SpRY system will allow researchers to efficiently modify C. albicans DNA that lacks NGG PAM sequences. IMPORTANCE Genetic modification of the human fungal pathogen Candida albicans allows us to better understand how fungi differ from humans at the molecular level and play essential roles in the development of therapeutics. CRISPR/Cas9-mediated genome editing systems can be used to introduce site-specific mutations to C. albicans. However, wild-type Cas9 is limited by the requirement of an NGG PAM site. CRISPR/SpRY targets a variety of different PAM sequences. We modified the C. albicans CRISPR/Cas9 system using the CRISPR/SpRY as a guide. We tested CRISPR/SpRY on C. albicans ADE2 and show that our SpRY system can facilitate genome editing independent of an NGG PAM sequence, thus allowing the investigator to target AT-rich sequences. Our system will potentially enable mutation of the 125 C. albicans genes which have been previously untargetable with CRISPR/Cas9. Additionally, our system will allow for precise targeting of many genomic locations that lack NGG PAM sites.

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Dramatic Improvement of CRISPR/Cas9 Editing in Candida albicans by Increased Single Guide RNA Expression

mSphere ◽

10.1128/msphere.00385-16 ◽

2017 ◽

Vol 2 (2) ◽

Cited By ~ 33

Author(s):

Henry Ng ◽

Neta Dean

Keyword(s):

Candida Albicans ◽

Genome Editing ◽

Virulence Factors ◽

Fungal Pathogen ◽

Fungal Virulence ◽

Important Conclusion ◽

Content Type ◽

Guide Rna ◽

Improve Efficiency ◽

Human Fungal Pathogen

ABSTRACT Candida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248 ) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans. The clustered regularly interspaced short palindromic repeat system with CRISPR-associated protein 9 nuclease (CRISPR/Cas9) has emerged as a versatile tool for genome editing in Candida albicans. Mounting evidence from other model systems suggests that the intracellular levels of single guide RNA (sgRNA) limit the efficiency of Cas9-dependent DNA cleavage. Here, we tested this idea and describe a new means of sgRNA delivery that improves previously described methods by ~10-fold. The efficiency of Cas9/sgRNA-dependent cleavage and repair of a single-copy yeast enhanced monomeric red fluorescent protein (RFP) gene was measured as a function of various parameters that are hypothesized to affect sgRNA accumulation, including transcriptional and posttranscriptional processing. We analyzed different promoters (SNR52, ADH1, and tRNA), as well as different posttranscriptional RNA processing schemes that serve to generate or stabilize mature sgRNA with precise 5′ and 3′ ends. We compared the effects of flanking sgRNA with self-cleaving ribozymes or by tRNA, which is processed by endogenous RNases. These studies demonstrated that sgRNA flanked by a 5′ tRNA and transcribed by a strong RNA polymerase II ADH1 promoter increased Cas9-dependent RFP mutations by 10-fold. Examination of double-strand-break (DSB) repair in strains hemizygous for RFP demonstrated that both homology-directed and nonhomologous end-joining pathways were used to repair breaks. Together, these results support the model that gRNA expression can be rate limiting for efficient CRISPR/Cas mutagenesis in C. albicans. IMPORTANCE Candida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248 ) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans.

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An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans

mSphere ◽

10.1128/mspheredirect.00149-17 ◽

2017 ◽

Vol 2 (2) ◽

Cited By ~ 33

Author(s):

Namkha Nguyen ◽

Morgan M. F. Quail ◽

Aaron D. Hernday

Keyword(s):

Candida Albicans ◽

Genome Editing ◽

Fungal Pathogen ◽

Gene Knockout ◽

Strain Engineering ◽

Molecular Genetic Analysis ◽

Content Type ◽

Highly Efficient ◽

Discovery Research ◽

Gene Knockouts

ABSTRACT Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans. Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans. We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCE Candida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans. This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen.

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Pmt-Mediated O Mannosylation Stabilizes an Essential Component of the Secretory Apparatus, Sec20p, in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.3.5.1164-1168.2004 ◽

2004 ◽

Vol 3 (5) ◽

pp. 1164-1168 ◽

Cited By ~ 22

Author(s):

Yvonne Weber ◽

Stephan K.-H. Prill ◽

Joachim F. Ernst

Keyword(s):

Endoplasmic Reticulum ◽

Candida Albicans ◽

Fungal Pathogen ◽

Wild Type ◽

Rapid Degradation ◽

Vesicle Traffic ◽

Human Fungal Pathogen ◽

Low Levels ◽

Lumenal Domain ◽

Secretory Apparatus

ABSTRACT Sec20p is an essential endoplasmic reticulum (ER) membrane protein in yeasts, functioning as a tSNARE component in retrograde vesicle traffic. We show that Sec20p in the human fungal pathogen Candida albicans is extensively O mannosylated by protein mannosyltransferases (Pmt proteins). Surprisingly, Sec20p occurs at wild-type levels in a pmt6 mutant but at very low levels in pmt1 and pmt4 mutants and also after replacement of specific Ser/Thr residues in the lumenal domain of Sec20p. Pulse-chase experiments revealed rapid degradation of unmodified Sec20p (38.6 kDa) following its biosynthesis, while the stable O-glycosylated form (50 kDa) was not formed in a pmt1 mutant. These results suggest a novel function of O mannosylation in eukaryotes, in that modification by specific Pmt proteins will prevent degradation of ER-resident membrane proteins via ER-associated degradation or a proteasome-independent pathway.

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Protection from Systemic Candida albicans Infection by Inactivation of the Sts Phosphatases

Infection and Immunity ◽

10.1128/iai.02789-14 ◽

2014 ◽

Vol 83 (2) ◽

pp. 637-645 ◽

Cited By ~ 19

Author(s):

Shamoon Naseem ◽

David Frank ◽

James B. Konopka ◽

Nick Carpino

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Fungal Growth ◽

Systemic Infection ◽

Content Type ◽

Host Immune Responses ◽

Human Fungal Pathogen ◽

Rapid Induction ◽

Inflammatory Damage ◽

Functional Inactivation

The human fungal pathogenCandida albicanscauses invasive candidiasis, characterized by fatal organ failure due to disseminated fungal growth and inflammatory damage. Thesuppressor ofTCRsignaling 1 (Sts-1) and Sts-2 are two homologous phosphatases that negatively regulate signaling pathways in a number of hematopoietic cell lineages, including T lymphocytes, mast cells, and platelets. Functional inactivation of both Sts enzymes leads to profound resistance to systemic infection byC. albicans, such that greater than 80% of mice lacking Sts-1 and -2 survive a dose ofC. albicans(2.5 × 105CFU/mouse) that is uniformly lethal to wild-type mice within 10 days. Restriction of fungal growth within the kidney occurs by 24 h postinfection in the mutant mice. This occurs without induction of a hyperinflammatory response, as evidenced by the decreased presence of leukocytes and inflammatory cytokines that normally accompany the antifungal immune response. Instead, the absence of the Sts phosphatases leads to the rapid induction of a unique immunological environment within the kidney, as indicated by the early induction of a proinflammatory cytokine (CXL10). Mice lacking either Sts enzyme individually display an intermediate lethality phenotype. These observations identify an opportunity to optimize host immune responses toward a deadly fungal pathogen.

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Systematic truncations of chromosome 4 and their responses to antifungals in Candida albicans

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00197-0 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Wasim Uddin ◽

Darshan Dhabalia ◽

S. M. Udaya Prakash ◽

M. Anaul Kabir

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Resistant Mutant ◽

Wild Type ◽

Human Fungal Pathogen ◽

Zones Of Inhibition ◽

Life Threatening ◽

Fluconazole Resistant ◽

Diffusion Assay ◽

Type Strains

Abstract Background Candida albicans is an opportunistic human fungal pathogen responsible for superficial and systemic life-threatening infections. Treating these infections is challenging as many clinical isolates show increased drug resistance to antifungals. Chromosome (Chr) 4 monosomy was implicated in a fluconazole-resistant mutant. However, exposure to fluconazole adversely affects Candida cells and can generate numerous mutations. Hence, the present study aimed to truncate Chr4 and challenge the generated Candida strains to antifungals and evaluate their role in drug response. Results Herein, Chr4 was truncated in C. albicans using the telomere-mediated chromosomal truncation method. The resulting eight Candida strains carrying one truncated homolog of Chr4 were tested for response to multiple antifungals. The minimal inhibitory concentration (MIC) for these strains was determined against three classes of antifungals. The MIC values against fluconazole, amphotericin B, and caspofungin were closer to that of the wild type strain. Microdilution assay against fluconazole showed that the mutants and wild type strains had similar sensitivity to fluconazole. The disc diffusion assay against five azoles and two polyenes revealed that the zones of inhibition for all the eight strains were similar to those of the wild type. Thus, none of the generated strains showed any significant resistance to the tested antifungals. However, spot assay exhibited a reasonably high tolerance of a few generated strains with increasing concentrations of fluconazole. Conclusion This analysis suggested that Chr4 aneuploidy might not underlie drug resistance but rather drug tolerance in Candida albicans.

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Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes

mSphere ◽

10.1128/msphere.00379-17 ◽

2017 ◽

Vol 2 (5) ◽

Cited By ~ 3

Author(s):

Arielle Butts ◽

Christian DeJarnette ◽

Tracy L. Peters ◽

Josie E. Parker ◽

Morgan E. Kerns ◽

...

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Second Generation ◽

Active Compounds ◽

Whole Cell ◽

Competitive Fitness ◽

Content Type ◽

Human Fungal Pathogen ◽

Conventional Drug ◽

Physiologically Active Compounds

ABSTRACT Conventional drug screening typically employs either target-based or cell-based approaches. The first group rely on biochemical assays to detect modulators of a purified target. However, hits frequently lack drug-like characteristics such as membrane permeability and target specificity. Cell-based screens identify compounds that induce a desired phenotype, but the target is unknown, which severely restricts further development and optimization. To address these issues, we have developed a second-generation target-based whole-cell screening approach that incorporates the principles of both chemical genetics and competitive fitness, which enables the identification of target-specific and physiologically active compounds from a single screen. We have chosen to validate this approach using the important human fungal pathogen Candida albicans with the intention of pursuing novel antifungal targets. However, this approach is broadly applicable and is expected to dramatically reduce the time and resources required to progress from screening hit to lead compound. Traditional approaches to drug discovery are frustratingly inefficient and have several key limitations that severely constrain our capacity to rapidly identify and develop novel experimental therapeutics. To address this, we have devised a second-generation target-based whole-cell screening assay based on the principles of competitive fitness, which can rapidly identify target-specific and physiologically active compounds. Briefly, strains expressing high, intermediate, and low levels of a preselected target protein are constructed, tagged with spectrally distinct fluorescent proteins (FPs), and pooled. The pooled strains are then grown in the presence of various small molecules, and the relative growth of each strain within the mixed culture is compared by measuring the intensity of the corresponding FP tags. Chemical-induced population shifts indicate that the bioactivity of a small molecule is dependent upon the target protein’s abundance and thus establish a specific functional interaction. Here, we describe the molecular tools required to apply this technique in the prevalent human fungal pathogen Candida albicans and validate the approach using two well-characterized drug targets—lanosterol demethylase and dihydrofolate reductase. However, our approach, which we have termed target abundance-based fitness screening (TAFiS), should be applicable to a wide array of molecular targets and in essentially any genetically tractable microbe. IMPORTANCE Conventional drug screening typically employs either target-based or cell-based approaches. The first group relies on biochemical assays to detect modulators of a purified target. However, hits frequently lack drug-like characteristics such as membrane permeability and target specificity. Cell-based screens identify compounds that induce a desired phenotype, but the target is unknown, which severely restricts further development and optimization. To address these issues, we have developed a second-generation target-based whole-cell screening approach that incorporates the principles of both chemical genetics and competitive fitness, which enables the identification of target-specific and physiologically active compounds from a single screen. We have chosen to validate this approach using the important human fungal pathogen Candida albicans with the intention of pursuing novel antifungal targets. However, this approach is broadly applicable and is expected to dramatically reduce the time and resources required to progress from screening hit to lead compound.

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The 5′ Untranslated Region of theEFG1Transcript Promotes Its Translation To Regulate Hyphal Morphogenesis inCandida albicans

mSphere ◽

10.1128/msphere.00280-18 ◽

2018 ◽

Vol 3 (4) ◽

Cited By ~ 2

Author(s):

Prashant R. Desai ◽

Klaus Lengeler ◽

Mario Kapitan ◽

Silas Matthias Janßen ◽

Paula Alepuz ◽

...

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Untranslated Regions ◽

Regulatory Sequence ◽

Transcriptional Level ◽

Content Type ◽

Reading Frame ◽

Human Fungal Pathogen ◽

Hyphal Morphogenesis ◽

Incandida Albicans

ABSTRACTExtensive 5′ untranslated regions (UTR) are a hallmark of transcripts determining hyphal morphogenesis inCandida albicans. The major transcripts of theEFG1gene, which are responsible for cellular morphogenesis and metabolism, contain a 5′ UTR of up to 1,170 nucleotides (nt). Deletion analyses of the 5′ UTR revealed a 218-nt sequence that is required for production of the Efg1 protein and its functions in filamentation, without lowering the level and integrity of theEFG1transcript. Polysomal analyses revealed that the 218-nt 5′ UTR sequence is required for efficient translation of the Efg1 protein. Replacement of theEFG1open reading frame (ORF) by the heterologous reporter geneCaCBGlucconfirmed the positive regulatory importance of the identified 5′ UTR sequence. In contrast to other reported transcripts containing extensive 5′ UTR sequences, these results indicate the positive translational function of the 5′ UTR sequence in theEFG1transcript, which is observed in the context of the nativeEFG1promoter. It is proposed that the 5′ UTR recruits regulatory factors, possibly during emergence of the native transcript, which aid in translation of theEFG1transcript.IMPORTANCEMany of the virulence traits that makeCandida albicansan important human fungal pathogen are regulated on a transcriptional level. Here, we report an important regulatory contribution of translation, which is exerted by the extensive 5′ untranslated regulatory sequence (5′ UTR) of the transcript for the protein Efg1, which determines growth, metabolism, and filamentation in the fungus. The presence of the 5′ UTR is required for efficient translation of Efg1, to promote filamentation. Because transcripts for many relevant regulators contain extensive 5′ UTR sequences, it appears that the virulence ofC. albicansdepends on the combination of transcriptional and translational regulatory mechanisms.

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The SUMO protease Ulp2 regulates genome stability and drug resistance in the human fungal pathogen Candida albicans

10.1101/2021.12.06.471441 ◽

2021 ◽

Author(s):

Marzia Rizzo ◽

Natthapon Soisangwan ◽

Jan Soetaert ◽

Samuel Vega-Estevez ◽

Anna Selmecki ◽

...

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Fungal Pathogen ◽

Genome Instability ◽

Antifungal Drugs ◽

Antifungal Drug ◽

Genome Plasticity ◽

Sumo Protease ◽

Human Fungal Pathogen ◽

Life Threatening

AbstractStress-induced genome instability in microbial organisms is emerging as a critical regulatory mechanism for driving rapid and reversible adaption to drastic environmental changes. In Candida albicans, a human fungal pathogen that causes life-threatening infections, genome plasticity confers increased virulence and antifungal drug resistance. Discovering the mechanisms regulating C. albicans genome plasticity is a priority to understand how this and other microbial pathogens establish life-threatening infections and develop resistance to antifungal drugs. We identified the SUMO protease Ulp2 as a critical regulator of C. albicans genome integrity through genetic screening. Deletion of ULP2 leads to hypersensitivity to genotoxic agents and increased genome instability. This increased genome diversity causes reduced fitness under standard laboratory growth conditions but enhances adaptation to stress, making ulp2Δ/Δ cells more likely to thrive in the presence of antifungal drugs. Whole-genome sequencing indicates that ulp2Δ/Δ cells counteract antifungal drug-induced stress by developing segmental aneuploidies of chromosome R and chromosome I. We demonstrate that intrachromosomal repetitive elements drive the formation of complex novel genotypes with adaptive power.

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Aneuploidy Underlies Tolerance and Cross-Tolerance to Drugs in Candida parapsilosis

Microbiology Spectrum ◽

10.1128/spectrum.00508-21 ◽

2021 ◽

Author(s):

Feng Yang ◽

Hui Lu ◽

Hao Wu ◽

Ting Fang ◽

Judith Berman ◽

...

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Candida Parapsilosis ◽

Cross Tolerance ◽

Content Type ◽

Human Fungal Pathogen

Candida parapsilosis is an emerging major human fungal pathogen, especially in neonates. Aneuploidy, having uneven numbers of chromosomes, is a well-known mechanism for adapting to stress in Candida albicans , the most common human fungal pathogen.

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CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Journal of Visualized Experiments ◽

10.3791/58764 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ben A. Evans ◽

Ethan S. Pickerill ◽

Valmik K. Vyas ◽

Douglas A. Bernstein

Keyword(s):

Candida Albicans ◽

Genome Editing ◽

Fungal Pathogen ◽

Human Fungal Pathogen

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