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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 labor...
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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.
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Some studies of active learning methods suggest that they are effective teaching tools, whereas others have found them to be no better than traditional lecture methods. The situation is much like the one that began to play out in ...
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Some studies of active learning methods suggest that they are effective teaching tools, whereas others have found them to be no better than traditional lecture methods. The situation is much like the one that began to play out in the 1950s with respect to the effects of psychotherapy. In that realm, it eventually became clear that the question "does therapy work?" was not the right one. It was more important to ask "which therapies result in clinically significant benefits when delivered by whom in what manner to which clients with what problems and how durable are the benefits"? This article suggests that it is time for researchers in the scholarship of teaching and learning to go beyond asking whether active learning "works" and address instead of a set of deeper questions about it. Doing so will require a more systematic and critical analysis of existing evidence as well as a new generation of research designed specifically to fill in the gaps in our understanding of what active learning methods can and cannot do.
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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 transcriptomewide quantitative mapping o...
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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 transcriptomewide 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 sitespecific 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.
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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 organis...
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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.
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RecQ DNA helicases function in DNA replication, recombination and repair. Although the precise cellular roles played by this family of enzymes remain elusive, the importance of RecQ proteins is clear; mutations in any of three hum...
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RecQ DNA helicases function in DNA replication, recombination and repair. Although the precise cellular roles played by this family of enzymes remain elusive, the importance of RecQ proteins is clear; mutations in any of three human RecQ genes lead to genomic instability and cancer. In this report, proteolysis is used to define a two‐domain structure for Escherichia coli RecQ, revealing a large (～59 kDa) N‐terminal and a small (～9 kDa) C‐terminal domain. A short N‐terminal segment (7 or 21 residues) is also shown to be sensitive to proteases. The effects of removing these regions of RecQ are tested in vitro. Removing 21 N‐terminal residues from RecQ severely diminishes its DNA‐dependent ATPase and helicase activities, but does not affect its ability to bind DNA in electrophoretic mobility shift assays. In contrast, removing the ～9 kDa C‐terminal domain from RecQ results in a fragment with normal levels of ATPase and helicase activity, but that has lost the ability to stably associate with DNA. These results establish the biochemical roles of an N‐terminal sequence motif in RecQ catalytic function and for the C‐terminal RecQ domain in stable DNA binding.
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RecQ DNA helicases function in DNA replication, recombination and repair. Although the precise cellular roles played by this family of enzymes remain elusive, the importance of RecQ proteins is clear; mutations in any of three hum...
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RecQ DNA helicases function in DNA replication, recombination and repair. Although the precise cellular roles played by this family of enzymes remain elusive, the importance of RecQ proteins is clear; mutations in any of three human RecQ genes lead to genomic instability and cancer. In this report, proteolysis is used to define a two-domain structure for Escherichia coli RecQ, revealing a large (～59 kDa) N-terminal and a small (～9 kDa) C-terminal domain. A short N-terminal segment (7 or 21 residues) is also shown to be sensitive to proteases. The effects of removing these regions of RecQ are tested in vitro. Removing 21 N-terminal residues from RecQ severely diminisches its DNA-dependent ATPase and helicase activities, but does not affect its ability to bind DNA in electrophoretic mobility shift assays. In contrast, removing the ～9 kDa C-terminal domain from RecQ results in a fragment with normal levels of ATPase and helicase activity, but that has lost the ability to stably associate with DNA. These results establish the biochemical roles of an N-terminal sequence motif in RecQ catalytic function and for the C-terminal RecQ domain in stable DNA binding.
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Like most other college and university students, most introductory psychology students—especially nonmajors—tend to forget the specifics of what they learned surprisingly soon after the course ends. Further, the typical introduc...
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Like most other college and university students, most introductory psychology students—especially nonmajors—tend to forget the specifics of what they learned surprisingly soon after the course ends. Further, the typical introduction to psychology course does surprisingly little to permanently alter many of the long-held misconceptions about human behavior and mental processes that many students bring to the course. In this article—originally presented in January 2016 at the 38th National Institute on The Teaching of Psychology—I review research on these depressing trends and propose that the best way to reverse them is to transform introductory psychology into a course that focuses entirely on combating potentially harmful misconceptions. I present a topic outline for the transformed course and describe some specific ways to organize and teach it that promote both active learning and critical thinking. I conclude by describing a set of research strategies that could be used nationwide to assess both the short- and long-term impact of this new entry-level psychology course compared with that of the more traditional format.
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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, w...
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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 (CaDcri). CaDcri is capable of generating siRNAs in vitro and is required for siRNA generation in vivo. In addition, CaDCRI complements a Dicer knockout in Saccharomyces castellii. restoring RNAi-mediated gene repression. Unexpectedly, deletion of the C. albicans CaDCRI results in a severe slow-growth pheno-type, whereas deletion of another core component of the RNAi pathway (CaAGOl) has little effect on growth, suggesting that CaDCRI may have an essential function in addition to producing siRNAs. Indeed CaDcri, 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.
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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-indep...
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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.
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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 the...
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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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