ABSTRACT Recent breakdowns in the supply chains of antiretroviral therapy (ART) to lower income countries, where they are needed most, underpin the pressing need for an accessible and scalable HIV cure that would allow ART-free control of the virus. The ‘shock-and-kill’ cure concept shows promise in its ability to reactivate HIV-1 transcription in latently infected cells in vivo in people living with HIV-1 but has failed to result in a meaningful reduction of the size of the reactivatable viral reservoir. We therefore hypothesised that the efficiency of reversal of transcriptional quiescence, and the resulting HIV-1 antigen expression and presentation, may be insufficient to achieve full immunological visibility of HIV-1-infected cells. To gain a deeper understanding of potential LRA-specific shortcomings, we developed a novel model of HIV-1 latency - Jurkat E6.1 subclonal cell lines, each harbouring a single, full-length, NL4.3-based provirus with a GFP OPT reporter inserted into the V5 loop of the viral Env protein. Using this model, we quantified HIV-1 reactivation, as well as expression and surface presentation of Env after treatment with a panel of known latency reversal agents (LRAs) and their synergistically-acting combinations. HIV-1 reactivation with the PKC agonist Bryostatin-1 limited the cell-surface presentation of viral Env, a phenomenon we found being linked to Bryostatin-1’s ability to induce the expression of the restriction factor GBP5 in T-cell lines and primary CD4+ T-cells, and induced a cellular state which was resistant to apoptosis. A combined treatment of Bryostatin-1 and the BET inhibitor (iBET) JQ1 resulted in synergistic HIV-1 reactivation and boosted levels of cell-surface Env, but failed to reduce resistance to apoptosis. In contrast, the combination of the SMAC mimetic (SMACm) AZD5582 and JQ1 markedly boosted cell-surface Env levels, without co-induction of GBP5, T-cell activation or apoptotic resistance. Crucially, HIV-1 Nef was found to be a potent antagonist of the cytotoxic killing of reactivating cells, most likely by its ability to inhibit apoptosis. Nef knockout, however, when paired with the AZD/JQ1 combination, displayed highest potency in inducing immune-mediated elimination of latently infected T-cells and presents a promising new approach for HIV cure programs.

The identification of novel antimalarials with activity against both the liver and blood stages of the parasite lifecycle would have the dual benefit of prophylactic and curative potential. However, one challenge of leveraging chemical hits from phenotypic screens is subsequent target identification. Here, we use in vitro evolution of resistance to investigate nine compounds from the Tres Cantos Antimalarial Set (TCAMS) with dual liver and asexual blood stage activity. We succeeded in eliciting resistance to four compounds, yielding mutations in acetyl CoA synthetase (AcAS), cytoplasmic isoleucine tRNA synthetase (cIRS), and protein kinase G (PKG) respectively. Using a combination of CRISPR editing and in vitro activity assays with recombinant proteins, we validate these as targets for TCMDC-125075 (AcAS), TCMDC-124602 (cIRS), and TCMDC-141334 and TCDMC-140674 (PKG). Notably, for the latter two compounds, we obtained a T618I mutation in the gatekeeper residue of PKG, consistent with direct interaction with the active site, which we modelled with molecular docking. Finally, we performed cross-resistance evaluation of the remaining five resistance-refractory compounds using the Antimalarial Resistome Barcode sequencing assay (AReBar), which examined a pool of 52 barcoded lines with mutations covering >30 common modes of action. None of the five compounds where in vitro evolution of resistance was not successful yielded validated hits using AReBar, indicating they likely act via novel mechanisms and may be candidates for further exploration.

Here we present pCASKD, a single-plasmid system for scarless chromosomal editing in Escherichia coli . Our plasmid pCASKD integrates CRISPR-Cas9-mediated counterselection, Lambda-Red recombineering, and temperature-sensitive plasmid curing into a 12 Kb vector to enable kilobase-scale insertions and deletions using a linear dsDNA donor and homologous recombination. Using the flagellar stator motAB locus as a model, we demonstrate that pCASKD enables efficient knock-in and knock-out edits with lower donor DNA input and reduced false positives compared with its parent, multi-plasmid system, No-SCAR. Using a single plasmid reduces transformation steps, accelerates screening, and increases the frequency of correctly edited clones. The protocol can be completed in five days, with potential for further optimization, offering a compact and efficient alternative for microbial genome engineering.

ABSTRACT Site-directed RNA editing, especially RNA base editing, allows for specific manipulation of RNA sequences, making it a useful approach for the correction of pathogenic mutations. Correction of RNA transcripts allows therapeutic gene editing in a safe and reversible manner and avoids permanent alterations in the genome. RNA-targeting CRISPR-Cas nucleases (e.g., CRISPR-Cas13) enable delivery within a single adeno-associated virus (AAV) vector for RNA base editing, making the approach clinically feasible. Here, we used the inactive CRISPR-Cas13bt3 (also known as Cas13X.1) fused to the ADAR2 deaminase domain (ADAR2DD) for targeted correction of inherited retinal disease (IRD) mutations. First, we show in vitro that dCas13bt3-ADAR2DD can efficiently correct a pathogenic nonsense mutation (c.130C>T [p.R44X]) found in the mouse Rpe65 gene and recover protein expression in retinal pigment epithelium cells (RPEs). Across clinically reported RPE65 mutations, we observed editing efficiencies ranging from 0% to 60%. In the Rpe65 -deficient mouse model of retinal degeneration (rd12), we observed that RNA base editing can recover Rpe65 expression in RPEs and rescue retinal function with no observable adverse effects. We further employed our RNA base editor against the large USH2A gene to assess the promise of RNA base editing for addressing untreatable IRDs caused by genes too large for AAV gene delivery. Against the human USH2A in vitro , we observed up to 60% on-target efficiency. We further found that gRNA mismatches, domain-inlaid ADAR2DD design and nucleocytoplasmic shuttling of the RNA base editor optimised on-target and bystander editing for a highly precise base editor. Against the mouse Ush2a in vitro , we similarly observed up to 60% on-target editing in mammalian cells, while in the Ush2a W3947X mice, we observed ∼12% on-target editing, with no impact on retinal structure or function, or transcriptome-wide editing. Overall, our findings demonstrate dCas13bt3-ADAR2DD as a potent tool for gene therapy against IRDs, addressing a significant unmet clinical need in ophthalmology.

Some phages have evolved the ability to cooperate to evade the immunity triggered by their bacterial host. A first exposure to the phage may weaken the host defences and allow later infections to be successful. Because this cooperation requires sequential infections, the phage can invade the host population only if its initial density is sufficiently high in a well-mixed environment. However, most natural bacterial populations are spatially structured. Could spatial structure create more favourable conditions for viral spread? Here we study the effect of spatial structure on the dynamics of cooperative anti-CRISPR (Acr) phages spreading in a population of CRISPR-Cas resistant Pseudomonas aeruginosa bacteria. We show experimentally that spatial structure does not always promote the spread of Acr-phages. In particular, the effect of spatial structure is modulated by the efficacy of the bacterial host’s CRISPR-Cas resistance and by the efficacy of the phage Acr protein. These results are discussed in the light of a mathematical model we developed to describe the spread of the phage. The model allows us to understand the ambivalent effects of spatial structure via its effects on the reproduction and on the persistence of the phage. More generally, we find that spatial structure can have opposite effects on the epidemiological dynamics of the phage, depending on the properties of the Acr protein encoded by the phage. This joint experimental and theoretical work yields a deeper understanding of the spatial dynamics of cooperative strategies in phages.

Understanding the gene regulatory mechanisms underlying brain function is crucial for advancing knowledge of the genetic basis of neurologic diseases. Cis-regulatory elements (CREs) play a pivotal role in gene regulation, and their evolutionary conservation can offer valuable insights. Importantly, the function and evolution of CREs are affected not only by primary sequence, but also by the cis - and trans -regulatory context. However, comparative functional analyses across species have been limited, leaving how these regulatory landscapes evolve in the brain largely unresolved. Here, we generated single-nucleus multiomic (snRNA- and snATAC-seq) data from cortex tissue across nine mammalian species and identified candidate CREs (cCREs) in a cell type–specific manner. We developed a multidimensional framework of conservation to assess sites of shared function that integrates sequence, chromatin accessibility, and enhancer–gene associations. Using massively parallel reporter assays (MPRA) in human neural progenitor cells and neurons, we measured activity of cCREs including both conserved and human-specific regions. CRISPR interference validated conserved enhancer function, including at neurodevelopmentally important genes like FAM181B . Motif enrichment identified transcription factors distinguishing conserved versus recent cCREs. Linkage disequilibrium score regression indicated that both conserved and human-specific cCREs were enriched for neuropsychiatric GWAS risk, while neurodegenerative risk was confined to conserved elements. Our findings define functional dimensions of enhancer conservation and demonstrate how regulatory evolution shapes human brain biology and disease susceptibility.

Genetic interactions are typically studied by looking at the phenotype that results from disruption of pairs of genes, as well as from higher order combinations of perturbations. Systematically interrogating all pairwise combinations provides insights into how genes are organized into pathways and complexes to sustain cellular homeostasis and how interacting genes respond to stressors and external signals. Genetic interactions have been studied extensively in yeast, due, in part, to the availability of a systematic collection of gene knockouts, and the development of Synthetic Genetic Array (SGA) technology. In contrast, such approaches are more challenging in human cells and therefore comparable data for human cells is scarce. This study introduces an innovative approach to functionally characterize genetic interactions in human cells through CRISPR/Cas9 screens using a pooled genome-wide knockout library in NALM6 cells. By combining a single guide RNA (sgRNA) targeting the gene of interest (aka the query) in cells already infected with an inducible genome-wide sgRNA pool, it is possible to achieve near saturation of genome-wide double knockouts. We conducted 26 of these screens, which we term “gene by genome-wide” knockout screens. This approach can be rapidly performed, in part, because it bypasses the need to generate genotyped isogenic knockout clones. Data from these screens identified both expected and novel synthetic lethal and synthetic rescue interactions, demonstrating that this strategy is effective for large-scale genetic research in human cells. Additionally, we show that these GBGW screens can be combined with chemical perturbation to reveal new synthetic interactions that are not apparent without drug treatment. Finally, we show that cDNA overexpression can be incorporated with genome-wide knockouts to systematically explore gain-of-function scenarios. The complete dataset is accessible on the ChemoGenix website (URL: https://chemogenix.iric.ca ).

Human papillomavirus (HPV) is a major etiological factor in cervical, anal, and oro-pharyngeal cancers. Although prophylactic vaccines have substantially reduced infec-tion rates, effective therapeutic options for established HPV-associated malignancies remain limited. This review provides an up-to-date overview of emerging strategies to treat HPV-driven tumors. Key approaches include immune checkpoint inhibitors, therapeutic vaccines such as VGX‑3100 and PRGN‑2012, and gene-editing tools like CRISPR/Cas9. Epigenetic drugs, particularly histone deacetylase inhibitors, show promise in reactivating silenced tumor suppressor genes and enhancing antitumor immunity. In addition, natural bioactive compounds and plant-derived molecules are being explored as complementary anti-HPV agents, while drug repurposing and combination therapies offer cost-effective opportunities to broaden treatment options. We also highlight the role of patient-derived organoid models as powerful platforms for personalized drug screening and functional assessment. By integrating these therapeutic innovations with precision oncology approaches, this review outlines a multi-dimensional framework aimed at improving clinical outcomes and quality of life for patients with HPV-associated cancers.

Terpenoids, vital pharmaceutical compounds, face production challenges due to low yields in native plants and ecological concerns. This review synthesizes recent advances in metabolic engineering strategies implemented across three complementary platforms: native medicinal plants, microbial systems, and heterologous plant hosts. We elucidate how the "Genomic Insights to Biotechnological Applications" paradigm, empowered by multi-omics technologies such as genomics, transcriptomics, metabolomics, etc., drives research advancements. These technologies facilitate the identification of key biosynthetic genes and regulatory networks. CRISPR-based tools, enzyme engineering, and subcellular targeting are highlighted as transformative strategies. Significant yield improvements have been demonstrated, with artemisinin and paclitaxel precursors showing considerable increases in production through strategic co-expression and optimization techniques. Persistent challenges such as metabolic flux balancing, cytotoxicity, and scale-up economics are discussed alongside emerging solutions including machine learning and photoautotrophic chassis. We conclude by outlining a roadmap for industrial translation that emphasizes the critical integration of systems biology and synthetic biology approaches to accelerate the transition of terpenoid biomanufacturing from discovery to commercial scale.

Genome editing technologies including CRISPR/Cas9, TALENs, and ZFNs offer a unique opportunity to eradicate HPV by directly disrupting its oncogenes E6 and E7, thereby restoring tumour‑suppressor pathways. In this review, we provide a comprehensive overview of recent advances in HPV‑targeted genome editing, summarising key preclinical findings that demonstrate tumour regression and apoptosis in HPV‑positive models, as well as the first‑in‑human clinical trials assessing safety and feasibility of local CRISPR‑based therapies. We also compare the relative strengths and limitations of each editing platform, discuss delivery strategies, and highlight their potential integration with immunotherapy and standard cancer treatments. While genome editing shows unprecedented precision and durability in targeting viral oncogenes, challenges such as efficient delivery, minimising off‑target effects, and navigating regulatory and ethical considerations remain. Continued optimisation of high‑fidelity nucleases, tissue‑specific delivery vehicles, and personalised guide design will be essential to translate these promising approaches into routine oncology practice. Genome editing thus represents a paradigm shift in HPV therapy, with the potential to transform management of both persistent infections and established cancers.

DNASE1L3 is a key endonuclease, essential for proper fragmentation and clearance of cell-free DNA (cfDNA). The p.R206C common variant impairs DNASE1L3 secretion and activity, causing aberrant cfDNA fragmentation and therefore affecting liquid biopsy-based screening and diagnostics. Existing studies on DNASE1L3 relied on resource-intensive murine models or plasmid-based overexpression, which do not accurately represent native expression. To address this, we developed an isogenic HEK293T cell line model by using CRISPR Prime Editing for endogenous expression of DNASE1L3 R206C . We analyzed the cfDNA composition directly from conditioned culture medium and found that fragment size distributions in mutant cells mimics the hypofragmented profiles previously observed in plasma samples from p.R206C carriers. We also showed that in vitro treatment of hypofragmented cfDNA with recombinant wildtype DNASE1L3 could enrich for mononucleosomal fragments, with fragment end-motifs characteristic of DNASE1L3 cleavage activity. This could open avenues for DNASE1L3 as a candidate pre-treatment agent to improve the accuracy and efficiency of cfDNA sequencing-based diagnostics in hypofragmented liquid biopsies. These findings demonstrate that our isogenic cell line model provides a controlled system to study cfDNA fragmentation biology and DNASE1L3 function.

Long-term recurrence in breast cancer is driven by reactivation of dormant disseminated tumour cells (DTCs) and remains a major clinical challenge, particularly in oestrogen receptor positive (ER⁺) tumours. This process is underpinned by regulation of the cell cycle machinery that controls quiescence maintenance and exit. HES1, a Notch pathway transcription factor, regulates key cell cycle genes and has been shown to demonstrate protein expression oscillations which are crucial to its function. Here, we sought to establish whether HES1 oscillations may regulate ER+ cancer cell quiescence and reactivation. To investigate this, we developed a fundamental in vitro model of cell cycle arrest and re-entry based on reversible CDK4/6 inhibition (CDK4/6i), compatible with quantitative single-cell live-imaging of an endogenous HES1 reporter. Consistent with earlier findings, HES1 exhibited ∼24-hour protein oscillations in cycling cells demonstrating a reproducible dip in protein expression prior to G1/S. During CDK4/6i-mediated arrest the ∼24h HES1 oscillation was lost, HES1 levels were maintained at a moderately higher level and HES1 exhibited smaller dips. Similar changes were observed in unperturbed, spontaneously quiescent cells. Following release from CDK4/6i and cell cycle re-entry, these alterations were reversed and the characteristic G1/S HES1 dip was observed. Preventing this dip at the point of release, by inducibly sustaining HES1 with a Tet-On system, impeded cell cycle re-entry and induced cell death. These findings suggest that manipulating HES1 dynamics could represent a promising therapeutic approach to prevent reactivation of dormant tumour cells. Significance Statement Breast cancer can recur years after initial treatment due to reactivation of dormant tumour cells. Understanding how these cells exit dormancy is crucial for preventing relapse. We investigated HES1, a transcription factor with rhythmic protein oscillations, and its role in regulating quiescence in oestrogen receptor-positive (ER⁺) breast cancer cells. Using live-cell imaging and a reversible cell cycle arrest model, we show that HES1 dynamics change during dormancy and reactivation, and that disrupting these oscillations prevents cell cycle re-entry and induces cell death. These findings reveal HES1 protein dynamics as a potential therapeutic vulnerability and highlight a novel strategy to target dormant cancer cells to prevent their reactivation.

CRISPR-Cas9 has revolutionized plant genome editing by enabling precise introduction of insertion/deletion (indel) mutations, critical for functional genomics and crop improvement studies. Sanger sequencing, combined with bioinformatics tools like the INDIGO webserver from Gear Genomics, is essential for validating these mutations. However, manual analysis of large numbers of Sanger sequencing (.ab1) files is labor-intensive, particularly when analyzing multiple guide RNA (gRNA) target regions. We developed a Python-based automation pipeline using Selenium with integrated highlighting of protospacer adjacent motif (PAM) regions in the resulting HTML reports. This pipeline enhances scalability of Sanger sequence data analysis and improves result interpretability by automating PAM region identification and supporting multiple gRNA regions. This tool significantly accelerates CRISPR-Cas9-mediated mutation analysis, offering a high throughput, reproducible solution for genome editing research.

Proteolysis-Targeting Chimeras (PROTACs) and Molecular Glue Degraders (MGDs) canonically target proteins for degradation by recruiting them to a single E3 ligase complex. While heterotrivalent PROTACs that can co-opt multiple E3 ligase complexes have been described, to our knowledge all MGDs reported to date are dependent on a single E3. Here, using orthogonal genetic screening, biophysical and structural analyses, we show that a monovalent MGD can covalently recruit CUL4DCAF16 and CRL1FBXO22 in a parallel and redundant manner to degrade SMARCA2/4. Deep mutational scanning identifies a single cysteine (Cys173) in DCAF16 essential for degrader activity, and intact protein MS confirms covalent adduct at this site. The cryo-EM structure of the DCAF16:SMARCA2:degrader ternary complex reveals a unique binding mode and a distinct interface of neo-interactions, providing insights into degrader specificity. We demonstrate that E3 ligase dependency can be tuned both chemically and genetically. Minimal alterations to the compound's "degradation tail" switches ligase preference from DCAF16 to FBXO22, while a single L59W mutation on DCAF16 is sufficient to drive DCAF16 engagement for otherwise FBXO22-dependent compounds. These results establish a molecular and structural framework for the design of tuneable dual glue degraders that could mitigate challenges from resistance mechanisms in degrader therapies.

ABSTRACT Efficient utilization of complex biomass-derived sugars and tolerance to inhibitors are key requirements for the viability of lignocellulosic-based biorefineries. In this study, a two-stage evolution of an industrial yeast strain engineered with a xylose isomerase pathway yielded strain AceY.14, which exhibited improved fermentative performance and increased tolerance to acetic acid. Whole-genome sequencing of the evolved strain identified SNPs in ZWF1 , a component of the pentose phosphate pathway (PPP), and in the G1 cyclin gene CLN3 , both of which were functionally validated through CRISPR and reverse engineering. The zwf1 E191D mutation reduced xylitol accumulation, alleviating inhibition of xylose isomerase and enhancing flux through the non-oxidative branch of the PPP, while the frameshift cln3 T556fs mutation unexpectedly improved acetic acid tolerance and xylose consumption in the evolved strain, also affecting cell size and growth. Genome sequencing of AceY.14 also revealed a significant reduction in the xylA gene copy number, likely decreasing the metabolic burden associated with high xylose isomerase expression. A synergistic effect was observed in the isu1 Δ /zwf1 Δ double mutant, further boosting xylose consumption rates. A diploid derivative (AceY-2n) demonstrated high productivity and robustness in fermentations using hydrolysates from various lignocellulosic feedstocks, highlighting the strain’s potential for industrial-scale applications. These findings reveal novel metabolic targets for strain optimization and offer valuable insights for the rational engineering of yeast platforms for sustainable biofuel and bioproduct production.

SUMMARY During development, many cell types transition from canonical to non-canonical cell cycles, such as endomitosis and endoreplication, in which they duplicate their DNA but do not divide, giving rise to polyploidy. Little is known on the regulation of endomitosis, where cells enter M phase, but do not perform cytokinesis. Here, we investigate how cells initiate and execute endomitosis in the C. elegans intestine and find that endomitotic cells fail to assemble a central spindle or initiate cytokinetic furrowing. We find that endomitotic cells transcriptionally repress multiple cytokinesis regulators, especially the central spindle factors ZEN-4 Mklp 1 , CYK-4 RacGap 1 and SPD-1 Prc 1 . Intestinal cells lose the capacity to perform cytokinesis in late embryogenesis, and we find that the conserved DREAM (DP, RB-related, E2F and MuvB) complex is involved in the repression of central spindle genes. Together, our work demonstrates that the transition to endomitosis is a well-defined switch that relies on the transcriptional repression of cytokinesis genes.

Engineering animal models with self-sustained luminescence could enable non-invasive longitudinal monitoring of molecular events in living animals. To create animal models that report physiology with autoluminescence, both luciferin biosynthesis enzymes and the luciferase need to be optimised. Previous work on engineering the autoluminescence pathway from fungi resulted in the development of nnLuz_v3, a version of Neonothopanus nambi luciferase with enhanced thermal stability. Here, we generated an nnLuz_v3 reporter of endogenous Cyp1a1 expression as a measure of aryl hydrocarbon receptor (AHR) activation, assessing the performance of nnLuz_v3 in vivo at physiologically relevant expression levels. As AHR dynamically responds to metabolic, environmental and dietary changes it provides a validated platform to assess novel luminescence approaches. In Cyp1a1-nnLuz mice bioluminescence signal was stable, allowing the generation of well-resolved luminescence images both on standard in vivo imaging equipment and consumer-grade cameras. Using mice and nematode models, we demonstrated limited oral availability of the fungal luciferin, potentially compatible with delivering the substrate via food or the microbiome. Our results are an encouraging first step in the generation of an autoluminescent mammalian model of a molecular event and encourage optimisation of other enzymes of the fungal luciferase pathway.

Progranulin, the precursor protein to seven and a half distinct granulin motifs (GRNs), has been implicated in a broad range of diseases. Progranulin depletion is one of the most frequent causes for hereditary Frontotemporal Dementia (FTD). On the other hand, elevated progranulin levels have been associated with increased malignancy of many tumours, manifesting in increased cell proliferation, migration, metastasis formation, and reduced sensitivity to chemotherapeutics. While some functions can be unambiguously attributed to either full-length progranulin or one or multiple of the different GRNs, much about the interplay between progranulin and GRNs remains unknown. Here, we aimed to test the effect of progranulin overexpression on cell-based tumorigenicity assays, assessing proliferation, migration, and colony formation, using the hepatocellular carcinoma cell line HepG2 and the glioblastoma cell line U87. We transduced these cells with lentiviral vectors to overexpress full-length progranulin, two different C-terminally truncated progranulin proteins, lacking either the last two or the last four GRNs, or a triple FLAG-tagged maltose binding protein as a control. We observed increased colony formation in HepG2 overexpressing the full-length progranulin but not the C-terminally truncated constructs. The U87 cell lines were neither affected by an increase in progranulin levels nor by the depletion of progranulin.

ABSTRACT KRAS mutations are among the most prevalent oncogenic drivers in non-small cell lung cancer (NSCLC), yet the mechanisms of therapeutic resistance to KRAS inhibitors in these cancers remains poorly understood. Here, we deploy high-throughput CRISPR base editing screens to systematically map resistance mutations to three mechanistically distinct KRAS-targeted therapies, including KRAS-G12C(OFF) inhibitor (adagrasib), RAS(ON) G12C-selective tri-complex inhibitor (RMC-4998), and RAS(ON) multi-selective tri-complex inhibitor (RMC-7977). Using both a saturation Kras tiling approach and cancer-associated mutation library, we identify common and compound-selective second-site resistance mutations in Kras , as well as gain-of-function and loss-of-function variants across cancer-associated genes that rewire signaling networks in a context-dependent manner. Notably, we identify a recurrent missense mutation in capicua ( Cic ), that promotes resistance to RMC-7977 in vitro and in vivo. Moreover, we show that targeting NFκB signaling in CIC-mutant cells can resensitize them to RAS pathway inhibition and overcome resistance.

Summary Asymmetric localization of mRNAs is a prevalent mechanism for spatial control of protein function and typically involves active transport by cytoskeletal motors. The mechanisms of recognition of localizing mRNAs by motor complexes are poorly understood. Egalitarian is an adaptor protein that binds localization signals in specific RNAs in Drosophila and recruits them to the dynein-dynactin complex for microtubule-based transport. We determined the crystal structure of Egalitarian in complex with the localization signal of the K10 mRNA. Three structural units of Egalitarian, a 3’-5’exonuclease domain, a linker and a C-terminal domain form shape-specific, base-directed and backbone interactions with the RNA. Based on the structure we identified conserved residues recognizing RNA in vitro . Genome-edited flies with mutations in these residues have deficits in Egalitarian function that are congruent with changes in in vitro RNA binding affinities. Our work demonstrates how a minimal RNA localization signal is recognized by an RNA localization factor.

Background/Objectives Hepatoblastoma (HB) is the most common form of pediatric liver cancer, with the vast majority of these tumors evidence of mutation and/or deregulation of the oncogenic transcription factors β-catenin (B), YAP (Y) and NRF2 (N). HB research has been hampered by a paucity of established cell lines, particularly those bearing these molecular drivers. All combinations of B, Y and N (i.e. BY, BN, YN and BYN) are tumorigenic when over-expressed in murine livers but it has not been possible to establish cell lines from primary tumors. Recently, we found that concurrent Crispr-mediate targeting of the Cdkn2a tumor suppressor locus allows for such immor-talized cell lines to be generated with high fidelity. Methods We generated 5 immortalized cell lines from primary Cdkn2a -targeted BN and YN HBs and characterized their properties. Notably, 4 of the 5 retain their ability to grow as subcutaneous or pulmonary tumors in the immune-competent mice from which they originated. Most notably, when maintained under hypoxia conditions for as little as 2 days, BN cells reversibly up-regulated the expression of numerous endothelial cell (EC)-specific genes and ac-quired EC-like properties that benefited tumor growth. Conclusions The above approach is currently the only means by which HB cell lines with pre-selected, clinically relevant oncogenic drivers can be generated and the only ones that can be studied in immune-competent mice. Its generic nature should allow HB cell lines with other oncogenic drivers to be derived. A collection of such cell lines will be useful for studying tumor cell-EC trans-differentiation, interactions with the immune environment and drug sensitivities. Simple Summary Most hepatoblastomas (HB) are associated with aberrant expression of β-catenin (B), YAP (Y) and/or NRF2 (N) transcription factors and can be modeled in mice by over-expressing pairwise of triple combination of these. Virtually no human or murine HB cell lines exist that bear these mutations. We describe here an efficient way to generate cell lines from primary BN and YN tumors. Moreover, one of the BN lines shows a remarkable ability to trans-differentiate into endothelial cells under hypoxic conditions that may facilitate angiogenesis. These cell lines along with previousl-derived BN and BYN lines showed similar sensitivities to drugs commonly used to treat HB. Because the approach for cell line derivation we describe is quite general, it should allow for the generation of additional lines driven by less common factors. A collection of such permanent and well-characterized cell lines will facilitate studies that are difficult or impractical to perform in vivo .

Human SAGA is a 20-subunit complex that stimulates transcription and is essential for development. The most prominent addition to SAGA in metazoans compared to yeast is a 150kDa splicing-factor module (SPL). SPL is also a part of the U2snRNP but its role in SAGA is elusive, partially due to absence of high-resolution structural information regarding its incorporation into the complex. In yeast, subunit TAF5 and TAF6 of SAGA are shared with the general transcription factor TFIID. In metazoans, gene duplication created proteins that occur only in SAGA (TAF5L and TAF6L) or in TFIID (TAF5 and TAF6). What functions of SAGA benefit from this protein specialization is unclear. Here we report the structure of endogenous human SAGA purified via an affinity-ligand from cells that were not disturbed by any genomic engineering tools such as CRISPR-Cas9. Our work reveals the high-resolution structure of SPL and of the TAF6L HEAT repeat domain that provides the SPL with a docking surface. We elucidate how SPL and the HEAT repeats are incorporated into SAGA. We find multiple major differences between TAF6L/TAF5L and the canonical paralogues that are directly implicated in structural re-arrangements required to accommodate SPL. Furthermore, SPL binding to SAGA is very different and occupying much less interaction surface than to U2snRNP. However, the two cases still share similar sequences in a helix that is deeply inserted into the SPL. The seemingly weaker interaction of SPL with SAGA raises the possibility that SAGA serves to relay this module to the splicing machinery. Our structure also suggests mutations that could uncouple SPL from SAGA to further interrogate the role of this module.

In human cells, a subset of tRNA-encoding genes contain introns. These are removed by a non-canonical splicing pathway in which the tRNA splicing endonuclease complex catalyzes intron excision and the resulting exons are subsequently ligated by the tRNA-ligase complex (tRNA-LC). Although recent studies have provided insights into the process of intron removal, the molecular mechanisms underpinning tRNA ligation by tRNA-LC remain elusive. The tRNA-LC is a hetero-pentameric protein assembly consisting of Ashwin, CGI-99, FAM98B, the DEAD-box helicase DDX1 and the catalytic subunit RTCB/HSPC117. Using cryo-EM, we have determined an atomic-resolution reconstruction of human tRNA-LC. We find that CGI-99, DDX1 and FAM98B form an alpha-helical bundle that contacts RTCB via an interface located on the opposite side from the location of the ligase active site and tethers DDX1 to the tRNA-LC via its C-terminal helix. FAM98B and CGI-99 extensively interact in an intricately co-folded heterodimer that clamps Ashwin in a pincer-like structure. Interaction analysis using structure-based mutants of tRNA-LC subunits supports the overall architecture of the complex. Finally, we show that the paralogous proteins FAM98A and FAM98C underpin the assembly of compositionally distinct RTCB-containing complexes that lack Ashwin and may have distinct cellular functions. Together, our results provide new insights into the assembly and mechanism of the tRNA ligase complex, shedding light on its functions in tRNA biogenesis and beyond.

The autoimmune disease systemic lupus erythematosus (SLE) is associated with genetic variants in the X-linked gene CXORF21 , which encodes the protein TASL. TASL acts as an adaptor in the IRF5 pathway and is necessary for the phosphorylation of IRF5 in response to TLR7 or TLR9 stimulation. Here, we investigate the role of TASL in the humoral immune response, and in the development of lupus in the B6.MRL lpr murine model of SLE. We find that while TASL is dispensable for their development, it is required for the full activation of B cells via endolysosomal TLR stimulation, and consequent interferon signalling and inflammatory cytokine expression. Additionally, TASL is crucial for the emergence of age-associated B cells (ABCs), a B cell population derived from the extrafollicular response that increases with age and is expanded in autoimmune disease, and the production of IgG2c antibodies. We also find that deletion of TASL prevents the onset of autoimmunity in the genetically-determined B6.MRL lpr model of lupus.

Type 1 diabetes can be cured by β–cell replacement in principle, yet recurrent autoimmunity and transplantation barriers rapidly destroy implanted cells. Genome–wide CRISPR screening by Cai et al . highlighted RNLS and HIVEP2 as candidate genes, but their value outside an autoimmune setting is unknown. Here, it was evaluated whether single-gene knockout of RNLS or HIVEP2 could similarly protect β-cell grafts against allo- and xenogeneic rejection. Murine β–TC–6 and human EndoC–βH1 cell lines were genetically edited using CRISPR-Cas9 to knockout RNLS or HIVEP2, and editing efficiencies were confirmed via T7 endonuclease I assay and TIDE analysis. Functional characterization indicated that RNLS deletion modestly impaired glucose-stimulated insulin secretion in murine cells, whereas HIVEP2 deletion showed no functional alterations in either cell line. For in vivo assessment, genetically edited β-cell spheroids were subcutaneously transplanted into CD-1 mice to model allo- (murine β-cells) and xenogeneic (human β-cells) rejection scenarios. Bioluminescence imaging revealed no protective effects of RNLS or HIVEP2 deletion, with grafts from both knockout groups displaying identical rejection kinetics compared to controls. These findings indicate that single-gene deletions of RNLS or HIVEP2 are insufficient for conferring meaningful protection against allo- or xenorejection, highlighting the necessity for combinatorial genome editing strategies or complementary biomaterial-based immunomodulation to achieve effective and sustained β-cell graft survival.