Biology subjects

Beisel, C.

Publications and source records attributed to Beisel, C..

At least 19 recordsLinked to original sources

bioRxiv2026

RNA editing and trans-splicing with reprogrammed tracrRNAs

Natural CRISPR-Cas9 systems rely on crRNA-tracrRNA duplexes to guide DNA targeting. Prior work showed that tracrRNAs could be reprogrammed to hybridize to cellular RNAs, resulting in their conversion into non-canonical crRNAs that enabled RNA detection and recording. However, the fate of the cellular RNA and the engineering opportunities it affords remain unexplored. Here, we show that the hybridized RNA is not inactivated, allowing the recruitment of Cas9 to the RNA duplex to drive RNA base editing and trans-splicing. Fusing ADAR2dd to dSpyCas9 and systematically engineering the reprogrammed tracrRNA (Rptr) enabled efficient and tunable A-to-I RNA editing, with on- and off-target profiles comparable to dCas13. The methodology extended to the compact CjeCas9 that could be further tailored for RNA targeting by deleting the HNH domain and mutating the PAM-interacting domain. Finally, utilizing Rptrs to block splicing enabled 3' and 5' RNA trans-splicing. Thus, Rptrs offer a versatile alternative to conventional Cas9 guide RNA architectures for programmable RNA manipulation.

Synthetic Biology↗

bioRxiv2026

A compact Druantia defense clears phage infections via single-stranded DNA recognition and directional duplex unwinding

Bacteria encode diverse anti-phage defense systems triggered by invader-specific molecular cues. Here, we report that the compact type III-A Druantia system recognizes exposed single-stranded DNA to drive phage clearance. Using representative systems from Escherichia coli, we show that the two encoded proteins, DruE and DruH, together clear restriction-sensitive or recombination-prone phages without affecting cell growth or viability. DruE dimerizes and engages DNA at exposed single-stranded regions to unwind DNA with 3'-to-5' directionality, resorting to unique molecular lock, wedge, and clamp elements that aid strand separation and processive translocation. DruH is a monomer in isolation and indirectly interacts with DruE and other host proteins under uninfected conditions, with an infection resulting in the dissociation of the complex. Taken together, our results reveal that exposed single-stranded DNA can trigger bacterial immunity through the directional helicase activity of type III-A Druantia. HighlightsO_LIThe E. coli Druantia III-A defense, comprising DruE and DruH, clears infecting phages C_LIO_LIDruE dimers bind exposed single-stranded DNA and unwind the upstream DNA duplex C_LIO_LI3'-to-5' DNA unwinding is aided by molecular lock, wedge, and clamp elements C_LIO_LIDruE interacts with DruH and host proteins, which are displaced upon infection C_LI

microbiology↗

bioRxiv2026

Dissecting multi-gene virulence phenotypes with base editing requires consideration of target-independent mutagenesis

Bacterial pathogenicity arises from complex genetic interactions that are difficult to characterise through single-gene deletions. CRISPR base editors can generate multiplexed gene knockouts, yet this technology remains unexplored for dissecting bacterial pathogenicity. Here, we developed a base-editing pipeline for multi-gene knockouts while revealing that target-independent editing can contribute to variability in clonal fitness. In the model pathogen Salmonella Typhimurium, we employed curable plasmids containing a cytidine deaminase base editor and a multi-spacer CRISPR array to introduce premature stop codons in up to nine genes encoding SPI-2 T3SS effector proteins. Target bases were efficiently edited, producing a multi-knockout strain that showed reduced virulence in vivo relative to single knockouts. However, whole-genome sequencing revealed off-target cytidine deaminase activity, which affected virulence in vivo in a clone-dependent manner. A statistical power analysis predicted how many edited mutants are needed to confidently measure fitness functions in the face of off-target mutations. Our work shows the potential and current limitations of multiplexed base editing in bacterial pathogens and highlights the need for properly addressing off-target mutations when deploying base editors to interrogate genotype-phenotype relationships.

microbiology↗

bioRxiv2026

The antiparasitics ivermectin and moxidectin trigger genomic and transcriptomic adaptation in bacteria

The macrocyclic lactone anthelmintics ivermectin and moxidectin are widely used for parasite control and show antibacterial activity in vitro. Given their structural relatedness to macrolide antibiotics, their broad use raises concerns about macrolide cross-resistance and altered bacterial physiology, yet the impact on gut bacterial isolates and underlying mechanisms remains poorly defined. Here, we combined Oxford Nanopore whole genome sequencing with Illumina RNA sequencing across six bacterial isolates analyzed as unexposed controls and as derivatives repeatedly exposed to ivermectin or moxidectin to identify genomic and transcriptomic signatures associated with exposure to these drugs. High-quality genome assemblies enabled integrated analyses across species with distinct cellular architectures and antimicrobial resistance gene arsenals. Across the panel, exposure was associated with i) widespread modulation of ribosome-linked genes and pathways, the molecular target of macrolide antibiotics; ii) context-dependent tuning of pre-existing antimicrobial resistance gene arsenals, such as macB and mdlB; and iii) recurrent alterations in potassium transport systems (ktrA and trkA). These findings suggest that repeated ivermectin or moxidectin exposure can reshape bacterial physiology in ways that may, in specific genetic backgrounds, co-modulate resistance-associated traits.

microbiology↗

bioRxiv2026

A type II Thoeris anti-phage defense enacts growth arrest independent of NAD depletion

Thoeris immune systems protect bacteria against invading phages through production of a signaling molecule by ThsB that activates the effector ThsA. Across the four defined types, the two most dominant (I and II) have been associated with abortive infection, with type I acting through the depletion of the essential coenzyme NAD+. Here, we show that a previously uncharacterized type II Thoeris system from Escherichia coli ATCC 25922 deviates from this paradigm. This system consists of the effector protein ThsA and two distinct signaling proteins ThsB1 and ThsB2. Heterologous expression of thsA and thsB2 confers anti-phage defense, while thsB1 is cytotoxic when expressed in common E. coli lab strains. Furthermore, while phage infection drives growth arrest, we could not detect any measurable decrease in NAD+ levels as well as standard markers of cell death. Together, these results suggest that Thoeris contains even greater functional diversity within the defined system types.

microbiology↗

bioRxiv2025

YprA family helicases provide the missing link between diverse prokaryotic immune systems

Bacteria and archaea possess an enormous variety of antivirus immune systems that often share homologous proteins and domains, some of which contribute to diverse defense strategies. YprA family helicases are central to widespread defense systems DISARM, Dpd, and Druantia. Here, through comprehensive phylogenetic and structural prediction analysis of the YprA family, we identify several major, previously unrecognized clades, with unique signatures of domain architecture and associations with other genes. Each YprA family clade defines a distinct class of defense systems, which we denote ARMADA (disARM-related Antiviral Defense Array), BRIGADE (Base hypermodification and Restriction Involving Genes encoding ARMADA-like and Dpd-like Effectors), or TALON (TOTE-like and ARMADA-Like Operon with Nuclease). In addition to the YprA-like helicase, ARMADA systems share two more proteins with DISARM. However, ARMADA YprA homologs are most similar to those of Druantia, suggesting ARMADA is a missing link connecting DISARM and Druantia. We show experimentally that ARMADA protects bacteria against a broad range of phages via a direct, non-abortive mechanism. We also discovered multiple families of satellite phage-like mobile genetic elements that often carry both ARMADA and Druantia Type III systems and show that these can provide synergistic resistance against diverse phages.

microbiology↗

bioRxiv2025

Hybrid-metagenomics reveal off-target effects of albendazole, ivermectin-albendazole and moxidectin-albendazole on the human gut bacteria.

The human whipworm parasite, Trichuris trichiura, poses a critical public health problem, affecting over 400 million people globally and responding poorly to the standard-of-care - benzimidazole chemotherapy. Recent efforts aimed at developing improved treatment options, the two macrolide-benzimidazole combination therapies ivermectin-albendazole and moxidectin-albendazole. Recent reports suggest that ivermectin and moxidectin possess antibacterial properties in vitro, hence there is a need to comprehensively characterize their off-target effects on the gut microbiome. In the framework of a randomized-controlled trial we collected stool samples of 204 T. trichiura-infected individuals in Cote dIvoire receiving albendazole (400mg), albendazole-ivermectin (400mg/200{micro}g/kg) and albendazole-moxidectin (400mg/8mg). Using a state-of-the-art hybrid sequencing approach that combines Illumina short read and Oxford Nanopore long read shotgun sequencing, we recovered over 800 high-quality metagenome-assembled genomes. Our analyses reveal that albendazole and albendazole-moxidectin induce taxonomic shifts in the gut microbiota with only mild functional consequences. In contrast, in individuals receiving higher quantities of albendazole-ivermectin (ivermectin > 12mg), based on their weight, we observed profoundly modulated taxonomic composition and microbial function, while the resistome was largely spared. These findings robustly confirm ivermectins antibacterial properties in the human gut, extending beyond previously reported in vitro effects. Given that ivermectin is a cornerstone in parasitic disease control, it is crucial to evaluate its broader impacts and considering more targeted, evidence-based treatment strategies.

genomics↗

bioRxiv2025

Comprehensive Analysis of CRISPR Array Repeat Mutations Reveals Subtype-Specific Patterns and Links to Spacer Dynamics

CRISPR-Cas systems are adaptive immune mechanisms in bacteria and archaea that protect against invading genetic elements by integrating short fragments of foreign DNA into CRISPR arrays. These arrays consist of repetitive sequences interspersed with unique spacers, guiding Cas proteins to recognize and degrade matching nucleic acids. The integrity of these repeat sequences is crucial for the proper function of CRISPR-Cas systems, yet their mutational dynamics remain poorly understood. In this study, we analyzed 56,343 CRISPR arrays across 25,628 diverse prokaryotic genomes to assess the mutation patterns in CRISPR array repeat sequences within and across different CRISPR subtypes. Our findings reveal, as expected to some extent, that mutation frequency is substantially higher in terminal repeat sequences compared to internal repeats consistently across system types. However, the mutation patterns exhibit an unexpected amount of variation among different CRISPR subtypes, suggesting that selective pressures and functional constraints shape repeat sequence evolution in distinct ways. Understanding these mutation dynamics provides insights into the stability and adaptability of CRISPR arrays across diverse bacterial and archaeal lineages. Additionally, we elucidate a novel relationship between repeat mutations and spacer dynamics, demonstrating that hotspots for terminal repeat mutations coincide with regions exhibiting spacer conservation. This observation corroborates recent findings by Fehrenbach et al. (2024) indicating that spacer deletions occur at a frequency 374 times greater than that of mutations and are significantly influenced by repeat misalignment. Our findings suggest that repeat mutations play a pivotal role in spacer retention or loss, or vice versa, thereby highlighting an evolutionary trade-off between the stability and adaptability of CRISPR arrays.

bioinformatics↗

bioRxiv2024

DemoTape: Computational demultiplexing of targeted single-cell sequencing data

BackgroundSingle-cell sequencing can provide novel insights into the understanding and treatment of diseases. In cancer, for example, intratumor heterogeneity is a major cause of treatment resistance and relapse. Although technological progress has substantially increased the throughput of sequenced cells, single-cell sequencing remains cost and labor-intensive. Multiplexing, i.e., the pooling and subsequent joint preparation and sequencing of samples, followed by a demultiplexing step, is a common practice to reduce expenses and confounding batch effects, especially in single-cell RNA sequencing. ResultsHere, we introduce demoTape, a computational demultiplexing method for targeted single-cell DNA sequencing (scDNA-seq) data based on a distance metric between individual cells at single-nucleotide polymorphisms loci. To validate demoTape, we sequence three B-cell lymphoma patients separately and multiplexed on the Tapestri platform. We find similar genotypes, clones, and evolutionary histories in all three samples when comparing the individual with the demultiplexed samples. Using the three individually sequenced samples, we simulate multiplexed ground truth data and show that demoTape outperforms state-of-the-art demultiplexing methods designed for RNA sequencing data. Additionally, we demonstrate through downsampling that the inferred clonal composition remained largely stable for samples with fewer cells despite the inevitable loss in resolution of low-frequency clones. ConclusionsMultiplexing and subsequent genotype-based demultiplexing of scDNA-seq will reduce costs and workload, eventually allowing the sequencing of more samples. This will open new possibilities and accelerate the investigation of biological questions where cellular heterogeneity on the genomic level plays a crucial role.

bioinformatics↗

bioRxiv2024

Large contribution of repeats to genetic variation in a transmission cluster of Mycobacterium tuberculosis

Repeats are the most diverse and dynamic, but also the least well understood component of microbial genomes. For all we know, repeat-associated mutations such as duplications, deletions, inversions, and gene conversion might be as common as point mutations, but because of short-read myopia and methodological bias they have received much less attention. Long-read sequencing opens the perspective of resolving repeats and systematically investigating the mutations they induce. For this study, we assembled the genomes of 16 closely related strains of the bacterial pathogen Mycobacterium tuberculosis from PacBio HiFi reads, with the aim of characterizing the full spectrum of DNA polymorphisms. We find that complete and accurate genomes can be assembled from HiFi reads, with read size being the main limitation in the presence of duplications. By combining a reference-free pangenome graph with extensive repeat annotation, we identified 110 variants, 58 of which can be assigned to repeat-associated mutational mechanisms such as strand slippage and homologous recombination. While recombination events are less frequent than point mutations, they can affect large regions and introduce multiple variants at once, as shown by three gene conversion events and a duplication of 7.3 kb that involve ppe18 and ppe57, two genes possibly involved in immune subversion. Our study shows that the contribution of repeat-associated mechanisms of mutation can be similar to that of point mutations at the microevolutionary scale of an outbreak. A large reservoir of unstudied genetic variation in this "monomorphic" bacterial pathogen awaits investigation.

genomics↗

bioRxiv2024

Cell-free expression with a quartz crystal microbalance enables rapid, dynamic, and label-free characterization of membrane-interacting proteins

Integral and interacting membrane proteins (IIMPs) constitute a vast family of biomolecules that perform essential functions in all forms of life. However, characterizing their interactions with lipid bilayers remains limited due to challenges in purifying and reconstituting IIMPs in vitro or labeling IIMPs without disrupting their function in vivo. Here, we report TXTL-QCMD to dynamically characterize interactions between diverse IIMPs and membranes without protein purification or labeling. As part of TXTL-QCMD, IIMPs are synthesized using cell-free transcription-translation (TXTL), and their interactions with supported lipid bilayers are measured using a quartz crystal microbalance with dissipation (QCMD). TXTL-QCMD reconstitutes known IIMP-membrane dependencies, including specific association with prokaryotic or eukaryotic membranes or oscillating interactions by the E. coli Min system. Applying TXTL-QCMD to the recently discovered Zorya anti-phage system unamenable to labeling, we discovered that ZorA and ZorB integrate together within the lipids found at the poles of bacteria while ZorE diffuses freely on the non-pole membrane. These efforts establish the potential of TXTL-QCMD to broadly characterize the large diversity of IIMPs.

biophysics↗

bioRxiv2024

MprF-mediated immune evasion is necessary for Lactiplantibacillus plantarum resilience in Drosophila gut during inflammation

BackgroundMultiple peptide resistance factor (MprF) confers resistance to cationic antimicrobial peptides (AMPs) in several pathogens, thereby enabling evasion of the host immune response. While MprF has been proven to be crucial for the virulence of various pathogens, its role in commensal gut bacteria remains uncharacterized. To close this knowledge gap, we used a common gut commensal of animals, Lactiplantibacillus plantarum, and its natural host, the fruit fly Drosophila melanogaster, as an experimental model to investigate the role of MprF in commensal-host interactions. ResultsThe L. plantarum {Delta}mprF mutant that we generated exhibited deficiency in the synthesis of lysyl-phosphatidylglycerol (Lys-PG), resulting in increased negative cell surface charge and increased susceptibility to AMPs. Susceptibility to AMPs had no effect on {Delta}mprF mutants ability to colonize guts of uninfected flies. However, we observed significantly reduced abundance of the {Delta}mprF mutant after infection-induced inflammation in the guts of wild-type flies but not flies lacking AMPs. These results demonstrate that host AMPs reduce the abundance of the {Delta}mprF mutant during infection. We found in addition that the {Delta}mprF mutant compared to wild-type L. plantarum induces a stronger intestinal immune response in flies due to the increased release of immunostimulatory peptidoglycan fragments, indicating an important role of MprF in promoting host tolerance to commensals. ConclusionOverall, our results demonstrate that MprF, besides its well-characterized role in pathogen immune evasion and virulence, is also an important resilience factor in maintaining stable microbiota-host interactions during intestinal inflammation.

microbiology↗

bioRxiv2023

An adapted method for Cas9-mediated editing reveals the species-specific role of β-glucoside utilization driving competition between Klebsiella species

Cas9-based gene editing tools have revolutionized genetics, enabling the fast and precise manipulation of diverse bacterial species. However, widely applicable genetic tools for non-model gut bacteria are unavailable. Here, we present a two-plasmid Cas9-based system designed for gene deletion and knock-in complementation in three members of the Klebsiella oxytoca species complex (KoSC), which we applied to study the genetic factors underlying the role of these bacteria in competition against Klebsiella pneumoniae. The system allowed efficient and precise editing via enhanced lambda Red expression and functionally-validated complementation with the use of universal bookmark targets in K. oxytoca, Klebsiella michiganensis, and Klebsiella grimontii. We revealed that the carbohydrate permease CasA is critical in ex vivo assays for K. pneumoniae inhibition by K. oxytoca but is neither sufficient nor required for K. michiganensis and K. grimontii. Thus, the adaptation of state-of-the-art genetic tools to KoSC allows the identification of species-specific functions in microbial competition. * ImportanceCas9-based gene editing tools have revolutionized bacterial genetics, yet, their application to non-model gut bacteria is frequently hampered by various limitations. We utilized a two-plasmid Cas9-based system designed for gene deletion in Klebsiella pneumoniae and demonstrate after optimization its utility for gene editing in three members of the Klebsiella oxytoca species complex (KoSC) namely K. oxytoca, K. michiganensis and K. grimontii. We then adapted a recently developed protocol for functional complementation based on universal bookmark targets applicable to all tested species. In summary, species specific adaptation of state-of-the-art genetic tools allow efficient gene deletion and complementation in types strains as well as natural isolates of KoSC members to study microbial interactions.

microbiology↗

bioRxiv2022

The protooncogene Ski regulates the neuron-glia switch during development of the mammalian cerebral cortex

The brain is the most complex organ in mammals and understanding the origin of this complexity is a major challenge for developmental biologists. Crucial to the size and morphology of the cortex is the timing and transition of neural stem cell (NSC) fate. An interesting candidate for modulating and fine tuning these processes is the transcriptional regulator Ski, a protooncogene expressed in cortical cells. Ski is involved in diverse cellular processes and epigenetic programs, and mice deficient in Ski exhibit complex central nervous system defects that resemble some of the features observed in patients with 1p36 deletion syndrome and Shprintzen-Goldberg syndrome. Here, we took advantage of in vivo transgenic labeling and next-generation sequencing to analyze the gene expression profiles of NSCs, basal progenitor (BP) cells, and newborn neurons (NBNs) from wildtype and Ski-deficient embryos throughout cortical development. We created a unique database that allowed us to identify and compare signaling pathways and transcriptional networks within each progenitor population in the presence and absence of Ski. We find that NSCs are the most affected cell population and uncover that mutant NSCs fail to switch to a gliogenic fate in time. We show that Ski functions in concert with the Bone Morphogenetic Protein (BMP) signaling pathway to alter the cell differentiation fate of NSCs from neurons to glia, which is key to generating adequate numbers of specific cell types during corticogenesis. Thus, by combining genetic tools and bioinformatic analysis, our work not only deepens the knowledge of how Ski functions in the brain, but also provides an immense resource for studying neurodevelopmental disorders.

systems biology↗

bioRxiv2022

Detection of isoforms and genomic alterations by high-throughput full-length single-cell RNA sequencing for personalized oncology

Understanding the complex background of cancer requires genotype-phenotype information in single-cell resolution. Here, we perform long-read single-cell RNA sequencing (scRNA-seq) on clinical samples from three ovarian cancer patients presenting with omental metastasis and increase the PacBio sequencing depth to 12,000 reads per cell. Our approach captures 152,000 isoforms, of which over 52,000 are novel. Isoform-level analysis accounting for non-coding isoforms reveals 20% overestimation of protein-coding gene expression on average. We also detect cell type-specific isoform and poly-adenylation site usage in tumor and mesothelial cells, and find that mesothelial cells transition into cancer-associated fibroblasts in the metastasis, partly through the TGF-{beta}/miR-29/Collagen axis. Furthermore, we identify gene fusions, including an experimentally validated IGF2BP2::TESPA1 fusion, which is misclassified as high TESPA1 expression in matched short-read data, and call mutations confirmed by targeted NGS cancer gene panel results. With these findings, we envision long-read scRNA-seq to become increasingly relevant in oncology and personalized medicine.

cancer biology↗

bioRxiv2022

Single-cell analysis reveals inter- and intratumour heterogeneity in metastatic breast cancer

Metastasis is the leading cause of cancer-related deaths of breast cancer patients. Some cancer cells in a tumour go through successive steps, referred to as the metastatic cascade, and give rise to metastases at a distant site. We know that the plasticity and heterogeneity of cancer cells play critical roles in metastasis but the precise underlying molecular mechanisms remain elusive. Here we aimed to identify molecular mechanisms of metastasis during colonization, one of the most important yet poorly understood steps of the cascade. We performed single-cell RNA-Seq (scRNA-Seq) on tumours and matched lung macrometastases of patient-derived xenografts of breast cancer. After correcting for confounding factors such as the cell cycle and the percentage of detected genes (PDG), we identified cells in three states in both tumours and metastases. Gene-set Enrichment Analysis revealed biological processes specific to proliferation and invasion in two states. Our findings suggest that these states are a balance between epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial transitions (MET) traits that results in so-called partial EMT phenotypes. Analysis of the top Differentially Expressed Genes (DEGs) between these cell states revealed a common set of partial EMT Transcriptions Factors (TFs) controlling gene expression, including ZNF750, OVOL2, TP63, TFAP2C and HEY2. Our data suggest that the TFs related to EMT delineate different cell states in tumours and metastases. The results highlight the marked interpatient heterogeneity of breast cancer but identify common features of single cells from five models of metastatic breast cancer.

cancer biology↗

bioRxiv2022

Improved prediction of bacterial CRISPRi guide efficiency through data integration and automated machine learning

CRISPR interference (CRISPRi), the targeting of a catalytically dead Cas protein to block transcription, is the leading technique to silence gene expression in bacteria. However, design rules for CRISPRi remain poorly defined, limiting predictable design for gene interrogation, pathway manipulation, and high-throughput screens. Here we develop a best-in-class prediction algorithm for guide silencing efficiency by systematically investigating factors influencing guide depletion in multiple genome-wide essentiality screens, with the surprising discovery that gene-specific features such as transcriptional activity substantially impact prediction of guide activity. Accounting for these features as part of algorithm development allowed us to develop a mixed-effect random forest regression model that provides better estimates of guide efficiency than existing methods, as demonstrated in an independent saturating screen. We further applied methods from explainable AI to extract interpretable design rules from the model, such as sequence preferences in the vicinity of the PAM distinct from those previously described for genome engineering applications. Our approach provides a blueprint for the development of predictive models for CRISPR technologies where only indirect measurements of guide activity are available.

bioinformatics↗

bioRxiv2022

gExcite - A start-to-end framework for single-cell gene expression, hashing, and antibody analysis

SummarySingle-cell RNA sequencing (scRNA-seq) based gene expression analysis is now an established powerful technique to decipher tissues at a single-cell level. Recently, CITE-seq emerged as a multimodal single-cell technology capturing gene expression and surface protein information from the same single-cells, which allows unprecedented insights into disease mechanisms and heterogeneity, as well as immune cell profiling. Multiple single-cell profiling methods exist, but they are typically focussed on either gene expression or antibody analysis, not their combination. Moreover, existing software suites are not easily scalable to a multitude of samples. To this end, we designed gExcite, a start-to-end workflow that provides both gene expression and CITE-seq analysis, as well as hashing deconvolution. Embedded in the Snakemake workflow manager, gExcite facilitates reproducible and scalable analyses. We showcase the output of gExcite on a study of different dissociation protocols on PBMC samples. AvailabilitygExcite is open source available on github at https://github.com/ETH-NEXUS/gExcite_pipeline The software is distributed under the GNU General Public License 3 (GPL3). Contactsinger@nexus.ethz.ch Supplementary InformationSupplementary information is available at the journals web site.

bioinformatics↗