• An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Infection.

      Schulte, Leon N; Schweinlin, Matthias; Westermann, Alexander J; Janga, Harshavardhan; Santos, Sara C; Appenzeller, Silke; Walles, Heike; Vogel, Jörg; Metzger, Marco; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (American Society for Microbiology (ASM), 2020-02-18)
      A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens.IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.
    • ANNOgesic: a Swiss army knife for the RNA-seq based annotation of bacterial/archaeal genomes.

      Yu, Sung-Huan; Vogel, Jörg; Förstner, Konrad U; HIRI, Helmoltz-Institut für RNA-basierteInfektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-09-01)
      To understand the gene regulation of an organism of interest, a comprehensive genome annotation is essential. While some features, such as coding sequences, can be computationally predicted with high accuracy based purely on the genomic sequence, others, such as promoter elements or noncoding RNAs, are harder to detect. RNA sequencing (RNA-seq) has proven to be an efficient method to identify these genomic features and to improve genome annotations. However, processing and integrating RNA-seq data in order to generate high-resolution annotations is challenging, time consuming, and requires numerous steps. We have constructed a powerful and modular tool called ANNOgesic that provides the required analyses and simplifies RNA-seq-based bacterial and archaeal genome annotation. It can integrate data from conventional RNA-seq and differential RNA-seq and predicts and annotates numerous features, including small noncoding RNAs, with high precision. The software is available under an open source license (ISCL) at https://pypi.org/project/ANNOgesic/.
    • Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA-Sequencing and Mass Cytometry.

      Winkels, Holger; Ehinger, Erik; Vassallo, Melanie; Buscher, Konrad; Dinh, Huy Q; Kobiyama, Kouji; Hamers, Anouk A J; Cochain, Clément; Vafadarnejad, Ehsan; Saliba, Antoine-Emmanuel; et al. (Amercan Heart Association, 2018-03-15)
      Using single-cell RNA-sequencing of aortic leukocytes from chow diet- and Western diet-fed Apoe-/- and Ldlr-/- mice, we detected 11 principal leukocyte clusters with distinct phenotypic and spatial characteristics while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on the single-cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic Apoe-/- and Ldlr-/- mice. We confirmed the phenotypic diversity of these clusters with a novel mass cytometry 35-marker panel with metal-labeled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of single-cell RNA-sequencing, mass cytometry, and fluorescence-activated cell sorting, we detected 3 principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Leukocyte cluster gene signatures revealed leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients.
    • Bacterial Adaptation to the Host's Diet Is a Key Evolutionary Force Shaping Drosophila-Lactobacillus Symbiosis.

      Martino, Maria Elena; Joncour, Pauline; Leenay, Ryan; Gervais, Hugo; Shah, Malay; Hughes, Sandrine; Gillet, Benjamin; Beisel, Chase; Leulier, François; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier, 2018-07-11)
      Animal-microbe facultative symbioses play a fundamental role in ecosystem and organismal health. Yet, due to the flexible nature of their association, the selection pressures that act on animals and their facultative symbionts remain elusive. Here we apply experimental evolution to Drosophila melanogaster associated with its growth-promoting symbiont Lactobacillus plantarum, representing a well-established model of facultative symbiosis. We find that the diet of the host, rather than the host itself, is a predominant driving force in the evolution of this symbiosis. Furthermore, we identify a mechanism resulting from the bacterium's adaptation to the diet, which confers growth benefits to the colonized host. Our study reveals that bacterial adaptation to the host's diet may be the foremost step in determining the evolutionary course of a facultative animal-microbe symbiosis.
    • Bacterial RNA Biology on a Genome Scale.

      Hör, Jens; Gorski, Stanislaw A; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-01-16)
      Bacteria are an exceedingly diverse group of organisms whose molecular exploration is experiencing a renaissance. While the classical view of bacterial gene expression was relatively simple, the emerging view is more complex, encompassing extensive post-transcriptional control involving riboswitches, RNA thermometers, and regulatory small RNAs (sRNAs) associated with the RNA-binding proteins CsrA, Hfq, and ProQ, as well as CRISPR/Cas systems that are programmed by RNAs. Moreover, increasing interest in members of the human microbiota and environmental microbial communities has highlighted the importance of understudied bacterial species with largely unknown transcriptome structures and RNA-based control mechanisms. Collectively, this creates a need for global RNA biology approaches that can rapidly and comprehensively analyze the RNA composition of a bacterium of interest. We review such approaches with a focus on RNA-seq as a versatile tool to investigate the different layers of gene expression in which RNA is made, processed, regulated, modified, translated, and turned over.
    • Barriers to genome editing with CRISPR in bacteria.

      Vento, Justin M; Crook, Nathan; Beisel, Chase L; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Springer, 2019-06-05)
      Genome editing is essential for probing genotype-phenotype relationships and for enhancing chemical production and phenotypic robustness in industrial bacteria. Currently, the most popular tools for genome editing couple recombineering with DNA cleavage by the CRISPR nuclease Cas9 from Streptococcus pyogenes. Although successful in some model strains, CRISPR-based genome editing has been slow to extend to the multitude of industrially relevant bacteria. In this review, we analyze existing barriers to implementing CRISPR-based editing across diverse bacterial species. We first compare the efficacy of current CRISPR-based editing strategies. Next, we discuss alternatives when the S. pyogenes Cas9 does not yield colonies. Finally, we describe different ways bacteria can evade editing and how elucidating these failure modes can improve CRISPR-based genome editing across strains. Together, this review highlights existing obstacles to CRISPR-based editing in bacteria and offers guidelines to help achieve and enhance editing in a wider range of bacterial species, including non-model strains.
    • The chlamydial deubiquitinase Cdu1 supports recruitment of Golgi vesicles to the inclusion.

      Auer, Daniela; Hügelschäffer, Sophie D; Fischer, Annette B; Rudel, Thomas; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Wiley, 2019-11-01)
      Chlamydia trachomatis is the main cause of sexually transmitted diseases worldwide. As obligate intracellular bacteria Chlamydia replicate in a membrane bound vacuole called inclusion and acquire nutrients for growth and replication from their host cells. However, like all intracellular bacteria, Chlamydia have to prevent eradication by the host's cell autonomous system. The chlamydial deubiquitinase Cdu1 is secreted into the inclusion membrane, facing the host cell cytosol where it deubiquitinates cellular proteins. Here we show that inactivation of Cdu1 causes a growth defect of C. trachomatis in primary cells. Moreover, ubiquitin and several autophagy receptors are recruited to the inclusion membrane of Cdu1-deficient Chlamydia. Interestingly, the growth defect of cdu1 mutants is not rescued when autophagy is prevented. We find reduced recruitment of Golgi vesicles to the inclusion of Cdu1 mutants indicating that vesicular trafficking is altered in bacteria without active deubiquitinase (DUB). Our work elucidates an important role of Cdu1 in the functional preservation of the chlamydial inclusion surface.
    • Competitive exclusion is a major bioprotective mechanism of lactobacilli against fungal spoilage in fermented milk products.

      Siedler, Solvej; Rau, Martin Holm; Bidstrup, Susanne; Vento, Justin M; Aunsbjerg, Stina Dissing; Bosma, Elleke F; McNair, Laura M; Beisel, Chase L; Neves, Ana Rute; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (American Society of Microbiology, 2020-01-31)
      A prominent feature of lactic acid bacteria (LAB) is their ability to inhibit growth of spoilage organisms in food, but hitherto research efforts to establish the mechanisms underlying bioactivity focused on the production of antimicrobial compounds by LAB. We show in this study, that competitive exclusion, i.e, competition for a limited resource by different organisms, is a major mechanism of fungal growth inhibition by lactobacilli in fermented dairy products. The depletion of the essential trace element manganese by two Lactobacillus species was uncovered as the main mechanism for growth inhibition of dairy spoilage yeast and molds. A manganese transporter (MntH1), representing one of the highest expressed gene products in both lactobacilli, facilitates the exhaustive manganese scavenging. Expression of the mntH1 gene was found to be strain-dependent, affected by species co-culturing and growth phase. Further, deletion of the mntH1 gene in one of the strains resulted in loss of bioactivity, proving this gene to be important for manganese depletion. The presence of a mntH gene displayed a distinct phylogenetic pattern within the Lactobacillus genus. Moreover, assaying the bioprotective ability in fermented milk of selected lactobacilli from ten major phylogenetic groups identified a correlation between the presence of mntH and bioprotective activity. Thus, manganese scavenging emerges as a common trait within the Lactobacillus genus, but differences in expression result in some strains showing more bioprotective effect than others.In summary, competitive exclusion through ion depletion is herein reported a novel mechanism in LAB to delay growth of spoilage contaminants in dairy products.IMPORTANCE In societies that have food choices, conscious consumers demand natural solutions to keep their food healthy and fresh during storage, simultaneously reducing food waste. The use of "good bacteria" to protect food against spoilage organisms has a long successful history, even though the molecular mechanisms are not fully understood. In this study, we show that depletion of free manganese is a major bioprotective mechanism of lactobacilli in dairy products. High manganese uptake and intracellular storage provides a link to the distinct non-enzymatic manganese catalyzed oxidative stress defense mechanism, previously described for certain lactobacilli. The evaluation of representative Lactobacillus species in our study identifies multiple relevant species groups for fungal growth inhibition via manganese depletion. Hence, through the natural mechanism of nutrient depletion, the use of dedicated bioprotective lactobacilli constitutes an attractive alternative to artificial preservation.
    • The conserved 3' UTR-derived small RNA NarS mediates mRNA crossregulation during nitrate respiration.

      Wang, Chuan; Chao, Yanjie; Matera, Gianluca; Gao, Qian; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Oxford Academic, 2019-12-21)
      Small noncoding RNAs (sRNAs) from mRNA 3' UTRs seem to present a previously unrecognized layer of bacterial post-transcriptional control whereby mRNAs influence each other's expression, independently of transcriptional control. Studies in Escherichia coli and Salmonella enterica showed that such sRNAs are natural products of RNase E-mediated mRNA decay and associate with major RNA-binding proteins (RBPs) such as Hfq and ProQ. If so, there must be additional sRNAs from mRNAs that accumulate only under specific physiological conditions. We test this prediction by characterizing candidate NarS that represents the 3' UTR of nitrate transporter NarK whose gene is silent during standard aerobic growth. We find that NarS acts by Hfq-dependent base pairing to repress the synthesis of the nitrite transporter, NirC, resulting in mRNA cross-regulation of nitrate and nitrite transporter genes. Interestingly, the NarS-mediated repression selectively targets the nirC cistron of the long nirBDC-cysG operon, an observation that we rationalize as a mechanism to protect the bacterial cytoplasm from excessive nitrite toxicity during anaerobic respiration with abundant nitrate. Our successful functional assignment of a 3' UTR sRNA from a non-standard growth condition supports the notion that mRNA crossregulation is more pervasive than currently appreciated.
    • CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9.

      Dugar, Gaurav; Leenay, Ryan T; Eisenbart, Sara K; Bischler, Thorsten; Aul, Belinda U; Beisel, Chase L; Sharma, Cynthia M; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier/ Cel Press, 2018-03-01)
      Cas9 nucleases naturally utilize CRISPR RNAs (crRNAs) to silence foreign double-stranded DNA. While recent work has shown that some Cas9 nucleases can also target RNA, RNA recognition has required nuclease modifications or accessory factors. Here, we show that the Campylobacter jejuni Cas9 (CjCas9) can bind and cleave complementary endogenous mRNAs in a crRNA-dependent manner. Approximately 100 transcripts co-immunoprecipitated with CjCas9 and generally can be subdivided through their base-pairing potential to the four crRNAs. A subset of these RNAs was cleaved around or within the predicted binding site. Mutational analyses revealed that RNA binding was crRNA and tracrRNA dependent and that target RNA cleavage required the CjCas9 HNH domain. We further observed that RNA cleavage was PAM independent, improved with greater complementarity between the crRNA and the RNA target, and was programmable in vitro. These findings suggest that C. jejuni Cas9 is a promiscuous nuclease that can coordinately target both DNA and RNA.
    • CRISPR-Cas Systems and the Paradox of Self-Targeting Spacers.

      Wimmer, Franziska; Beisel, Chase L; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Frontiers, 2019-01-01)
      CRISPR-Cas immune systems in bacteria and archaea record prior infections as spacers within each system's CRISPR arrays. Spacers are normally derived from invasive genetic material and direct the immune system to complementary targets as part of future infections. However, not all spacers appear to be derived from foreign genetic material and instead can originate from the host genome. Their presence poses a paradox, as self-targeting spacers would be expected to induce an autoimmune response and cell death. In this review, we discuss the known frequency of self-targeting spacers in natural CRISPR-Cas systems, how these spacers can be incorporated into CRISPR arrays, and how the host can evade lethal attack. We also discuss how self-targeting spacers can become the basis for alternative functions performed by CRISPR-Cas systems that extend beyond adaptive immunity. Overall, the acquisition of genome-targeting spacers poses a substantial risk but can aid in the host's evolution and potentially lead to or support new functionalities.
    • The CRISPR/Cas system in Neisseria meningitidis affects bacterial adhesion to human nasopharyngeal epithelial cells.

      Heidrich, Nadja; Hagmann, Antony; Bauriedl, Saskia; Vogel, Jörg; Schoen, Christoph; HIRI, Helmoltz-Institut für RNA-basierteInfektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-07-30)
      Neisseria meningitidis, a commensal β-proteobacterium of the human nasopharynx, constitutes a worldwide leading cause of sepsis and epidemic meningitis. A recent genome-wide association study suggested an association of its type II-C CRISPR/Cas system with carriage and thus less invasive lineages. Here, we show that knock-out strains lacking the Cas9 protein are impaired in the adhesion to human nasopharyngeal cells which constitutes a central step in the pathogenesis of invasive meningococcal disease. Transcriptome sequencing data further suggest that meningococcal Cas9 does not affect the expression of surface adhesins but rather exerts its effect on cell adhesion in an indirect manner. Consequently, we speculate that the meningococcal CRISPR/Cas system exerts novel functions beyond its established role in defence against foreign DNA.
    • CRP-cAMP mediates silencing of Salmonella virulence at the post-transcriptional level.

      El Mouali, Youssef; Gaviria-Cantin, Tania; Sánchez-Romero, María Antonia; Gibert, Marta; Westermann, Alexander J; Vogel, Jörg; Balsalobre, Carlos; HIRI, Helmoltz-Institut für RNA-basierteInfektionsforschung, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. (2018-01-01)
      Invasion of epithelial cells by Salmonella enterica requires expression of genes located in the pathogenicity island I (SPI-1). The expression of SPI-1 genes is very tightly regulated and activated only under specific conditions. Most studies have focused on the regulatory pathways that induce SPI-1 expression. Here, we describe a new regulatory circuit involving CRP-cAMP, a widely established metabolic regulator, in silencing of SPI-1 genes under non-permissive conditions. In CRP-cAMP-deficient strains we detected a strong upregulation of SPI-1 genes in the mid-logarithmic growth phase. Genetic analyses revealed that CRP-cAMP modulates the level of HilD, the master regulator of Salmonella invasion. This regulation occurs at the post-transcriptional level and requires the presence of a newly identified regulatory motif within the hilD 3'UTR. We further demonstrate that in Salmonella the Hfq-dependent sRNA Spot 42 is under the transcriptional repression of CRP-cAMP and, when this transcriptional repression is relieved, Spot 42 exerts a positive effect on hilD expression. In vivo and in vitro assays indicate that Spot 42 targets, through its unstructured region III, the 3'UTR of the hilD transcript. Together, our results highlight the biological relevance of the hilD 3'UTR as a hub for post-transcriptional control of Salmonella invasion gene expression.
    • A decade of advances in transposon-insertion sequencing.

      Cain, Amy K; Barquist, Lars; Goodman, Andrew L; Paulsen, Ian T; Parkhill, Julian; van Opijnen, Tim; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Springer Nature, 2020-06-12)
      It has been 10 years since the introduction of modern transposon-insertion sequencing (TIS) methods, which combine genome-wide transposon mutagenesis with high-throughput sequencing to estimate the fitness contribution or essentiality of each genetic component in a bacterial genome. Four TIS variations were published in 2009: transposon sequencing (Tn-Seq), transposon-directed insertion site sequencing (TraDIS), insertion sequencing (INSeq) and high-throughput insertion tracking by deep sequencing (HITS). TIS has since become an important tool for molecular microbiologists, being one of the few genome-wide techniques that directly links phenotype to genotype and ultimately can assign gene function. In this Review, we discuss the recent applications of TIS to answer overarching biological questions. We explore emerging and multidisciplinary methods that build on TIS, with an eye towards future applications.
    • A detailed cell-free transcription-translation-based assay to decipher CRISPR protospacer-adjacent motifs.

      Maxwell, Colin S; Jacobsen, Thomas; Marshall, Ryan; Noireaux, Vincent; Beisel, Chase L; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Elsevier, 2018-02-24)
      The RNA-guided nucleases derived from the CRISPR-Cas systems in bacteria and archaea have found numerous applications in biotechnology, including genome editing, imaging, and gene regulation. However, the discovery of novel Cas nucleases has outpaced their characterization and subsequent exploitation. A key step in characterizing Cas nucleases is determining which protospacer-adjacent motif (PAM) sequences they recognize. Here, we report advances to an in vitro method based on an E. coli cell-free transcription-translation system (TXTL) to rapidly elucidate PAMs recognized by Cas nucleases. The method obviates the need for cloning Cas nucleases or gRNAs, does not require the purification of protein or RNA, and can be performed in less than a day. To advance our previously published method, we incorporated an internal GFP cleavage control to assess the extent of library cleavage as well as Sanger sequencing of the cleaved library to assess PAM depletion prior to next-generation sequencing. We also detail the methods needed to construct all relevant DNA constructs, and how to troubleshoot the assay. We finally demonstrate the technique by determining PAM sequences recognized by the Neisseria meningitidis Cas9, revealing subtle sequence requirements of this highly specific PAM. The overall method offers a rapid means to identify PAMs recognized by diverse CRISPR nucleases, with the potential to greatly accelerate our ability to characterize and harness novel CRISPR nucleases across their many uses.
    • Distinct timescales of RNA regulators enable the construction of a genetic pulse generator.

      Westbrook, Alexandra; Tang, Xun; Marshall, Ryan; Maxwell, Colin S; Chappell, James; Agrawal, Deepak K; Dunlop, Mary J; Noireaux, Vincent; Beisel, Chase L; Lucks, Julius; et al. (Wiley-Blackwell, 2019-01-13)
      To build complex genetic networks with predictable behaviours, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher-order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely employed to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA-based transcriptional regulation, using small transcription activating RNAs (STARs) and CRISPR interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell-free transcription-translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ODE-based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modelling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity. This article is protected by copyright. All rights reserved.
    • Dual RNA-seq of Orientia tsutsugamushi informs on host-pathogen interactions for this neglected intracellular human pathogen.

      Mika-Gospodorz, Bozena; Giengkam, Suparat; Westermann, Alexander J; Wongsantichon, Jantana; Kion-Crosby, Willow; Chuenklin, Suthida; Wang, Loo Chien; Sunyakumthorn, Piyanate; Sobota, Radoslaw M; Subbian, Selvakumar; et al. (Nature Publishing Group, 2020-07-03)
      Studying emerging or neglected pathogens is often challenging due to insufficient information and absence of genetic tools. Dual RNA-seq provides insights into host-pathogen interactions, and is particularly informative for intracellular organisms. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium that causes the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning to study the transcriptional architecture of Ot, we find evidence for wide-spread post-transcriptional antisense regulation. Comparing the host response to two clinical isolates, we identify distinct immune response networks for each strain, leading to predictions of relative virulence that are validated in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.
    • An educational module to explore CRISPR technologies with a cell-free transcription-translation system

      Collias, Daphne; Marshall, Ryan; Collins, Scott P.; Beisel, Chase L.; Noireaux, Vincent; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (Oxford Academic, 2019-05-21)
      Within the last 6 years, CRISPR-Cas systems have transitioned from adaptive defense systems in bacteria and archaea to revolutionary genome-editing tools. The resulting CRISPR technologies have driven innovations for treating genetic diseases and eradicating human pests while raising societal questions about gene editing in human germline cells as well as crop plants. Bringing CRISPR into the classroom therefore offers a means to expose students to cutting edge technologies and to promote discussions about ethical questions at the intersection of science and society. However, working with these technologies in a classroom setting has been difficult because typical experiments rely on cellular systems such as bacteria or mammalian cells. We recently reported the use of an E. coli cell-free transcription-translation (TXTL) system that simplifies the demonstration and testing of CRISPR technologies with shorter experiments and limited equipment. Here, we describe three educational modules intended to expose undergraduate students to CRISPR technologies using TXTL. The three sequential modules comprise (i) designing the RNAs that guide DNA targeting, (ii) measuring DNA cleavage activity in TXTL and (iii) testing how mutations to the targeting sequence or RNA backbone impact DNA binding and cleavage. The modules include detailed protocols, questions for group discussions or individual evaluation, and lecture slides to introduce CRISPR and TXTL. We expect these modules to allow students to experience the power and promise of CRISPR technologies in the classroom and to engage with their instructor and peers about the opportunities and potential risks for society.
    • Einzelzell-RNA-Sequenzierung beleuchtet den Infektionsprozess

      Saliba, Antoine-Emmanuel; Westermann, Alexander J.; Vogel, Jörg; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Schneider-Straße 2, 97080 Würzburg. Germany. (2017-10-11)
    • Eleven grand challenges in single-cell data science.

      Lähnemann, David; Köster, Johannes; Szczurek, Ewa; McCarthy, Davis J; Hicks, Stephanie C; Robinson, Mark D; Vallejos, Catalina A; Campbell, Kieran R; Beerenwinkel, Niko; Mahfouz, Ahmed; et al. (BMC, 2020-02-07)
      The recent boom in microfluidics and combinatorial indexing strategies, combined with low sequencing costs, has empowered single-cell sequencing technology. Thousands-or even millions-of cells analyzed in a single experiment amount to a data revolution in single-cell biology and pose unique data science problems. Here, we outline eleven challenges that will be central to bringing this emerging field of single-cell data science forward. For each challenge, we highlight motivating research questions, review prior work, and formulate open problems. This compendium is for established researchers, newcomers, and students alike, highlighting interesting and rewarding problems for the coming years.