How advancements in molecular biology impact education and training.

Molecular biology, broadly defined as the investigation of complex biomolecules in the laboratory, is a rapidly advancing field and as such the technologies available to investigators are constantly evolving. This constant advancement has obvious advantages because it allows students and researchers to perform more complex experiments in shorter periods of time. One challenge with such a rapidly advancing field is that techniques that had been vital for students to learn how to perform are now not essential for a laboratory scientist. For example, while cloning a gene in the past could have led to a publication and form the bulk of a PhD thesis project, technology has now made this process only a step toward one of these larger goals and can, in many cases, be performed by a company or core facility. As teachers and mentors, it is imperative that we understand that the technologies we teach in the lab and classroom must also evolve to match these advancements. In this perspective, we discuss how the rapid advances in gene synthesis technologies are affecting curriculum and how our classrooms should evolve to ensure our lessons prepare students for the world in which they will do science.

Characterization of endoplasmic reticulum-associated degradation in the human fungal pathogen Candida albicans.

Background: Candida albicans is the most prevalent human fungal pathogen. In immunocompromised individuals, C. albicans can cause serious systemic disease, and patients infected with drug-resistant isolates have few treatment options. The ubiquitin-proteasome system has not been thoroughly characterized in C. albicans. Research from other organisms has shown ubiquitination is important for protein quality control and regulated protein degradation at the endoplasmic reticulum (ER) via ER-associated protein degradation (ERAD). Methods: Here we perform the first characterization, to our knowledge, of ERAD in a human fungal pathogen. We generated functional knockouts of C. albicans genes encoding three proteins predicted to play roles in ERAD, the ubiquitin ligases Hrd1 and Doa10 and the ubiquitin-conjugating enzyme Ubc7. We assessed the fitness of each mutant in the presence of proteotoxic stress, and we used quantitative tandem mass tag mass spectrometry to characterize proteomic alterations in yeast lacking each gene. Results: Consistent with a role in protein quality control, yeast lacking proteins thought to contribute to ERAD displayed hypersensitivity to proteotoxic stress. Furthermore, each mutant displayed distinct proteomic profiles, revealing potential physiological ERAD substrates, co-factors, and compensatory stress response factors. Among candidate ERAD substrates are enzymes contributing to ergosterol synthesis, a known therapeutic vulnerability of C. albicans. Together, our results provide the first description of ERAD function in C. albicans, and, to our knowledge, any pathogenic fungus.

CRISPR-Mediated Genome Editing in the Human Fungal Pathogen C. albicans.

Cas9-mediated genome editing is one tool investigators can use to study fungal pathogens. Such methodologies allow the investigator to examine how fungal cells differ from human cells and thus potentially identify novel therapeutic targets. In this chapter, we describe how CRISPR-mediated genome editing can be used to edit the genome of the most prevalent human fungal pathogen C. albicans. A cassette encoding a fungal optimized Cas9 nuclease and guide RNA is integrated into the C. albicans genome. The guide RNA targets Cas9 to the complementary genome sequence, and Cas9 cleaves the DNA. A repair template encoding whatever changes the investigator wished to make to the genome is co-transformed with the cassette and repairs the break via homologous recombination, thus introducing the change to the genome. The method we describe enables the researcher to edit the C. albicans genome and then efficiently remove the editing machinery and antibiotic resistance markers. This allows one to sequentially edit the C. albicans genome when multiple changes are desired. In addition, we provide notes that provide guidance on how the described protocols can be altered to meet the demands of the researcher. In these notes, we also describe the recent development of a more flexible CRISPR system that has a relaxed PAM site specificity. These and other advancements make CRISPR-mediated genome editing a practical approach when one needs to genetically alter C. albicans.

Stay-on-Task Exercises as a Tool To Maintain Focus during a CRISPR CURE.

Course-based undergraduate research experiences (CURE) offer the chance for students to experience authentic research investigation in a classroom setting. Such hands-on experiences afford unique opportunities work on a semi-independent research project in an efficient, structured environment. We have developed a CRISPR CURE in which undergraduate and graduate students use in silico, in vitro, and in vivo techniques to edit a fungal genome. During the development of this course, we have found that the asynchronous nature of the CRISPR CURE activities can be disruptive and lead to unproductive class time. To overcome this challenge, we have developed stay-on-task exercises (SOTEs). These short low-stakes assessments provide structured activities that are performed during these asynchronous incubation periods. SOTE activities leverage potentially unproductive class time and complement the CURE learning objectives. We have found SOTEs to be one method of maintaining classroom structure during a CURE. Furthermore, SOTE complexity, length, and subject can be easily modified to match course learning objectives.

CRISPR mediated genome editing, a tool to dissect RNA modification processes.

Though over 100 distinct RNA modifications have been identified, the roles for many of these modifications in vivo remain unknown. Genome editing is one tool investigators are using to better understand the roles these modifications play and the consequences of their absence. In this chapter, we describe how CRISPR mediated genome editing can be used to interrogate the process of RNA modification in C. albicans. Furthermore, we discuss how the protocols described can be altered to meet experimental demands. The underlying theory on which these protocols are based are applicable to a variety of model systems. The protocols described utilize the widely used S. pyogenes Cas9, but the field of genome editing is quickly evolving. We discuss the recent developments of more flexible CRISPR systems that can target a greater number of sites in the genome. These and other advancements make CRISPR mediated genome editing a practical methodology to investigate RNA modification.

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

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. albicansADE2 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. albicansADE2 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.

Pseudouridine synthase 7 impacts Candida albicans rRNA processing and morphological plasticity.

RNA can be modified in over 100 distinct ways, and these modifications are critical for function. Pseudouridine synthases catalyse pseudouridylation, one of the most prevalent RNA modifications. Pseudouridine synthase 7 modifies a variety of substrates in Saccharomyces cerevisiae including tRNA, rRNA, snRNA, and mRNA, but the substrates for other budding yeast Pus7 homologues are not known. We used CRISPR-mediated genome editing to disrupt Candida albicans PUS7 and find absence leads to defects in rRNA processing and a decrease in cell surface hydrophobicity. Furthermore, C. albicans Pus7 absence causes temperature sensitivity, defects in filamentation, altered sensitivity to antifungal drugs, and decreased virulence in a wax moth model. In addition, we find C. albicans Pus7 modifies tRNA residues, but does not modify a number of other S. cerevisiae Pus7 substrates. Our data suggests C. albicans Pus7 is important for fungal vigour and may play distinct biological roles than those ascribed to S. cerevisiae Pus7.

An Introduction to CRISPR-Mediated Genome Editing in Fungi.

Central dogma, transformation, and genome editing are key biological concepts for which junior scientists must gain experience during training. Here we present an exercise that introduces these concepts in a single practical laboratory exercise. Our exercise utilizes CRISPR/Cas9 genome editing to introduce a stop codon into Saccharomyces cerevisiae ADE2. This edit leads to the buildup of an adenine precursor that dyes the edited cells red. As the repair template, guide RNA, and Cas9 are all encoded in our vector, transformation can be performed in 2 hours. Furthermore, since all components of the Cas9/CRISPR system are encoded by the vector, specialized equipment and reagents, such as a PCR machine or oligonucleotides, are not required to perform the experiments as designed. As such, these exercises provide an efficient cost-effective introduction to a wide variety of key molecular biology concepts and lay the foundation for more rigorous investigations in upper-level classes and independent research projects.

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

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6.

Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the Candida albicans UME6 Zn2+-binding domain, a known regulator of C. albicans filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type C. albicans. Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest.

New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi.

We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans, Saccharomyces cerevisiae, Candida glabrata, and Naumovozyma castellii These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms.IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata, as well as in the related yeasts S. cerevisiae and N. castellii The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.

Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA.

Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function.

Protein interactions in genome maintenance as novel antibacterial targets.

Antibacterial compounds typically act by directly inhibiting essential bacterial enzyme activities. Although this general mechanism of action has fueled traditional antibiotic discovery efforts for decades, new antibiotic development has not kept pace with the emergence of drug resistant bacterial strains. These limitations have severely restricted the therapeutic tools available for treating bacterial infections. Here we test an alternative antibacterial lead-compound identification strategy in which essential protein-protein interactions are targeted rather than enzymatic activities. Bacterial single-stranded DNA-binding proteins (SSBs) form conserved protein interaction "hubs" that are essential for recruiting many DNA replication, recombination, and repair proteins to SSB/DNA nucleoprotein substrates. Three small molecules that block SSB/protein interactions are shown to have antibacterial activity against diverse bacterial species. Consistent with a model in which the compounds target multiple SSB/protein interactions, treatment of Bacillus subtilis cultures with the compounds leads to rapid inhibition of DNA replication and recombination, and ultimately to cell death. The compounds also have unanticipated effects on protein synthesis that could be due to a previously unknown role for SSB/protein interactions in translation or to off-target effects. Our results highlight the potential of targeting protein-protein interactions, particularly those that mediate genome maintenance, as a powerful approach for identifying new antibacterial compounds.

Identification of small molecules that disrupt SSB-protein interactions using a high-throughput screen.

Bacterial single-stranded DNA-binding proteins (SSBs) recruit a diverse array of genome maintenance enzymes to their sites of action through direct protein interactions. The essential nature of these SSB-protein interactions makes inhibitors that block SSB-partner complex formation valuable biochemical tools and attractive potential antibacterial agents. However, many of these protein-protein interactions are weak and not amenable to the high-throughput nature of small molecule screens. Here I describe a high-throughput screen to identify small molecules that inhibit the interaction between Exonuclease I (ExoI) and the final 10 amino acids of the SSB C-terminal tail (SSB-Ct). The strength of the binding between ExoI and the SSB-Ct tail is fundamental to the interaction's utility in the high-throughput screen.

Genes come and go: the evolutionarily plastic path of budding yeast RNase III enzymes.

Our recent finding that the Candida albicans RNase III enzyme CaDcr1 is an unusual, multifunctional RNase III coupled with data on the RNase III enzymes from other fungal species prompted us to seek a model that explained the evolution of RNase III's in modern budding yeast species. CaDcr1 has both dicer function (generates small RNA molecules from dsRNA precursors) and Rnt1 function, (catalyzes the maturation of 35S rRNA and U4 snRNA). Some budding yeast species have two distinct genes that encode these functions, a Dicer and RNT1, whereas others have only an RNT1 and no Dicer. As none of the budding yeast species has the canonical Dicer found in many other fungal lineages and most eukaryotes, the extant species must have evolved from an ancestor that lost the canonical Dicer, and evolved a novel Dicer from the essential RNT1 gene. No single, simple model could explain the evolution of RNase III enzymes from this ancestor because existing sequence data are consistent with two equally plausible models. The models share an architecture for RNase III evolution that involves gene duplication, loss, subfunctionalization, and neofunctionalization. This commentary explains our reasoning, and offers the prospect that further genomic data could further resolve the dilemma surrounding the budding yeast RNase III's evolution.

Ruler arrays reveal haploid genomic structural variation.

Despite the known relevance of genomic structural variants to pathogen behavior, cancer, development, and evolution, certain repeat based structural variants may evade detection by existing high-throughput techniques. Here, we present ruler arrays, a technique to detect genomic structural variants including insertions and deletions (indels), duplications, and translocations. A ruler array exploits DNA polymerase's processivity to detect physical distances between defined genomic sequences regardless of the intervening sequence. The method combines a sample preparation protocol, tiling genomic microarrays, and a new computational analysis. The analysis of ruler array data from two genomic samples enables the identification of structural variation between the samples. In an empirical test between two closely related haploid strains of yeast ruler arrays detected 78% of the structural variants larger than 100 bp.

Candida albicans Dicer (CaDcr1) is required for efficient ribosomal and spliceosomal RNA maturation.

The generation of mature functional RNAs from nascent transcripts requires the precise and coordinated action of numerous RNAs and proteins. One such protein family, the ribonuclease III (RNase III) endonucleases, includes Rnt1, which functions in fungal ribosome and spliceosome biogenesis, and Dicer, which generates the siRNAs of the RNAi pathway. The recent discovery of small RNAs in Candida albicans led us to investigate the function of C. albicans Dicer (CaDcr1). CaDcr1 is capable of generating siRNAs in vitro and is required for siRNA generation in vivo. In addition, CaDCR1 complements a Dicer knockout in Saccharomyces castellii, restoring RNAi-mediated gene repression. Unexpectedly, deletion of the C. albicans CaDCR1 results in a severe slow-growth phenotype, whereas deletion of another core component of the RNAi pathway (CaAGO1) has little effect on growth, suggesting that CaDCR1 may have an essential function in addition to producing siRNAs. Indeed CaDcr1, the sole functional RNase III enzyme in C. albicans, has additional functions: it is required for cleavage of the 3' external transcribed spacer from unprocessed pre-rRNA and for processing the 3' tail of snRNA U4. Our results suggest two models whereby the RNase III enzymes of a fungal ancestor, containing both a canonical Dicer and Rnt1, evolved through a series of gene-duplication and gene-loss events to generate the variety of RNase III enzymes found in modern-day budding yeasts.

Genotype to phenotype: a complex problem.

We generated a high-resolution whole-genome sequence and individually deleted 5100 genes in Sigma1278b, a Saccharomyces cerevisiae strain closely related to reference strain S288c. Similar to the variation between human individuals, Sigma1278b and S288c average 3.2 single-nucleotide polymorphisms per kilobase. A genome-wide comparison of deletion mutant phenotypes identified a subset of genes that were conditionally essential by strain, including 44 essential genes unique to Sigma1278b and 13 unique to S288c. Genetic analysis indicates the conditional phenotype was most often governed by complex genetic interactions, depending on multiple background-specific modifiers. Our comprehensive analysis suggests that the presence of a complex set of modifiers will often underlie the phenotypic differences between individuals.

Small-molecule tools for dissecting the roles of SSB/protein interactions in genome maintenance.

Bacterial single-stranded DNA-binding proteins (SSBs) help to recruit a diverse array of genome maintenance enzymes to their sites of action through direct protein interactions. For all cases examined to date, these interactions are mediated by the evolutionarily conserved C terminus of SSB (SSB-Ct). The essential nature of SSB protein interactions makes inhibitors that block SSB complex formation valuable biochemical tools and attractive potential antibacterial agents. Here, we identify four small molecules that disrupt complexes formed between Escherichia coli SSB and Exonuclease I (ExoI), a well-studied SSB-interacting enzyme. Each compound disrupts ExoI/SSB-Ct peptide complexes and abrogates SSB stimulation of ExoI nuclease activity. Structural and biochemical studies support a model for three of the compounds in which they compete with SSB for binding to ExoI. The fourth appears to rely on an allosteric mechanism to disrupt ExoI/SSB complexes. Subsets of the inhibitors block SSB-Ct complex formation with two other SSB-interaction partners as well, which highlights their utility as reagents for investigating the roles of SSB/protein interactions in diverse DNA replication, recombination, and repair reactions.