Understanding how cells mitigate lysosomal damage is critical for unraveling pathogenic mechanisms of lysosome-related diseases. Here we use organelle-specific proteomics in iPSC-derived neurons (i 3 Neuron) and an in vitro lysosome-damaging assay to demonstrate that lysosome damage, caused by the aggregation of Ceroid Lipofuscinosis Neuronal 4 (CLN4)-linked DNAJC5 mutants on lysosomal membranes, serves as a critical pathogenic linchpin in CLN4-associated neurodegeneration. Intriguingly, in non-neuronal cells, a ubiquitin-dependent microautophagy mechanism downregulates CLN4 aggregates to counteract CLN4-associated lysotoxicity. Genome-wide CRISPR screens identify the ubiquitin ligase CHIP as a central microautophagy regulator that confers ubiquitin-dependent lysosome protection. Importantly, CHIP’s lysosome protection function is transferrable, as ectopic CHIP improves lysosomal function in CLN4 i 3 Neurons, and effectively alleviates lipofuscin accumulation and neurodegeneration in a Drosophila CLN4 disease model. Our study establishes CHIP-mediated microautophagy as a key organelle damage guardian that preserves lysosome integrity, offering new insights into therapeutic development for CLN4 and other lysosome-related neurodegenerative diseases.

Recently, there has been a significant outbreak of clinical pneumonia caused by the Human Metapneumovirus, particularly in Northern regions of the People’s Republic of China, causing thousands of hospitalisation cases. Such an outbreak has grown fresh concerns with regards to a potential spread of the novel infectious disease to several other world countries and causation of public health-related difficulties that may be similar in nature with the effects of the SARS-CoV-2-induced COVID-19 pandemic, which occurred from March 2020 to March 2022 before the disease finally became endemic in nature. Throughout the COVID-19 pandemic, a novel immunological research narrative was developed, in which a wider inclusion of natural immunity-based elements was recommended as part of an update in general approaches contained by immunotherapeutic and vaccine-related clinical research. Particularly, it has been suggested that a fairly decreased concentration of Type I and Type III elements from the host interferon system be placed in the central area of the natural immunity-based immunisation and immunisation adjuvance. Several clinical trials have confirmed the important position of such interferon system elements in the natural immunity department responsible for immunising functions. Given the fact that major components of the natural immune system have recently shown to display considerable adaptive immunity-like traits, such as specificity and long-term “memory”, natural immunity-based vaccination may now be deemed as scientifically plausible, contrary to initial scientific projections that they can only constitute vaccine adjuvants. Approaches as such may include a low dose of Type I and Type III Interferon-, and perhaps protollin-based treatment of nasopharyngeal tissues, as well as of natural and adaptive lymphocytes, and of plasmacytoid dendritic cells also, which represent both factories for Type I and Type III Interferons, as well as valid immune system-based vaccine candidates against infectious and oncological diseases, alongside natural and adaptive lymphocytes. Such components of the immune system may be utilised in combination to confer the most effective version of such an overall candidate of a clinical response. Other vaccine candidates may involve live-attenuated viral genomes either lacking the interferon-suppressive genes or containing them as the only slightly active genetic regions, with the overall purpose of stimulating an evolutionary push of the interferon-encoding genes to outcompete the already advanced stages of microbial evolution, whose stronghold seems to be largely upon the host interferon system. Other approaches may also involve the development of live-attenuated pathogen-derived vaccines that have Interferon I and III-encoding genes inserted into the viral genome as its sole active genetic components. There may be a novel, experimental process involving the isolation of common cold-inducing viruses, such as Rhinoviral agents, during the beginning of local, seasonal outbreaks and perhaps inducing their weakening as well, in clinical laboratories that are located in multiple distinct geographical areas of the hemisphere where the fall season has begun, prior to the performance of a small-scale gene editing through the insertion of active Type I and Type III Interferon-encoding genes into the genome of such viruses, prior to their release back into the local environment, via the performance of CRISPR-Cas9. Specifically, microbial genes involved in the causation of pathogenesis and maintenance of pathophysiology would be substantially attenuated, and genes involved in microbial reproduction and transmission perhaps not as much. Such a process may turn such viruses and possibly also other microbes into spreadable vaccines, as the immune system would automatically be activated once viruses as such undergo receptor-mediated endocytosis and start expressing their genes. At least some of the genes encoding proteins that antagonise the host interferon system could be removed prior to the insertion of human genes encoding various elements of it, particularly in situations where microbial agents are known to antagonise it in a problematic manner, whether directly or indirectly. If an overall procedure as such is performed accurately and matches all bioethical guidelines, then at least only common cold diseases in the upper respiratory tract, including the Rhinovirus-induced disease, may be prevented in many cases and probably even gradually become eradicated in the end, given the fact that an automatic synthesis of Type I and Type III Interferons by pathogenic microbes could lead to a robust and proportional rate of immune sensitisation that would lead to their lysis and disposal, making it probable that even microbes that are normally causative of major clinical disease would be destroyed before they would be able to induce the first symptoms. Active genes encoding Pattern Recognition Receptor (PRR) Activators matching to the microbe could also be inserted into its genome, perhaps to restore normative levels of microbial sensing by the host, natural immune system. Perhaps, inhalers and injectable sera containing a fairly decreased dosage of such potential transmissible factories for Type I and Type III Interferons, and possibly also for specific Pattern Recognition Receptor Activator and/or Agonist proteins, as such may be prepared to fill in any remote gap to the production of a full, herd-immunity effect throughout human populations. Such a potential overall update in vaccine innovation and development could even impact the evolutionary trajectory of various single drug and multidrug antibiotic resistant pathogenic bacteria, which can represent additional, unnecessary burdens for patients with various viral infections.

Neurons are highly asymmetric post-mitotic cells whose processes extend long distances to facilitate communication. Consequently, they encounter the complex problem of maintaining their structure and function over long distances. Over the last decade, research into the components composing the axon of a neuron has revealed the presence of local machinery of protein synthesis and deployment within different parts of the axon. However, there is still a need to understand how the endogenous proteome and transcriptome within the axon are distributed. The last decade has also witnessed a growth of technology capable of specifically labeling proteins and molecules of RNA. They are primarily based on gene editing techniques and recombinant antibody technology. Advances in technology now enable the delivery of large payloads of genetic material, paving the way for an in-depth investigation into the endogenous processes occurring within the axons of adult neurons. These capabilities open up exciting opportunities to address critical questions, potentially leading to new insights and strategies for treating neurodegenerative diseases. The review discusses different techniques available to a neuroscientist to help answer questions concerning the localization and transport of molecules within the axon. For instance, CRISPR is used to make specific changes to the genome and provide a means to tag endogenous proteins. Using these advances, in theory, it is possible to label molecules at scale and elucidate the role of different compartments that support protein synthesis and their subsequent deployment to specific regions within the axon.

Colorectal cancer (CRC) represents the third-leading cause of cancer-related deaths. Knowledge covering diverse cellular and molecular data from individual patients has become valuable for diagnosis, prognosis, and treatment selection. Here, we present an in-depth comparative mRNA-seq and microRNA-seq analysis of tissue samples from 32 CRC, pairing tumors with adjacent healthy tissues. The differential expression gene (DEG) analysis revealed an interconnection between nutrients, metabolic programs, and cell cycle pathways. We focused on the impact of overexpressed SLC7A11 (xCT) and SLC3A2 genes which compose the cystine/glutamate transporter (Xc-) system. To assess the oncogenic potency of the Xc-system in a cellular setting, we applied a knowledge-based approach for analyzing gene perturbations from CRISPR screens across various cell types as well as using a variety of functional assays in five primary patient-derived organoid cell models to functionally verify our hypothesis. We identified a previously undescribed cell surface protein signature predicting chemotherapy resistance and further highlighted the causality and potential of pharmacological blockage of ferroptosis as promising avenue for cancer therapy. Biological processes such as redox homeostasis, ion/amino acid transporters and de novo nucleotide synthesis were associated with these co-dependent genes in patient specimens. This study highlighted a number of overlooked genes as potential clinical targets for CRC and promotes stem cell-based, patient-individual in vitro model systems as a versatile partner platform to functionally validate in silico predictions, with focus on SLC7A11 and its associated genes in tumorigenesis.

Eukaryotic phototrophs depend on the activity of two engines (the plastid and the mitochondrion) to generate the energy required for cellular metabolism. Because of their overlapping functions, both activities must be closely coordinated. At the plastid level, optimization occurs through alternative electron transport, the diversion of excess electrons from the linear transport chain, and metabolic exchanges. A similar process takes place in the mitochondria, with documented evidence of energy and redox equivalents being exchanged between the two organelles. Organelle-organelle energy interactions at the physiological level are well established in diatoms, an ecologically significant member of phytoplankton. Yet the molecular components involved in this process remain largely unknown. Here, we identify a Mitochondrial Carrier Family (MCF) transporter, MCFc, located at the plastid envelope of Phaeodactylum tricornutum , which seems to be widely distributed in complex algae. We then compare the performance of a wild-type and a mutant lacking MCFc. An analysis of spectroscopic and oxygen exchange data unveiled altered energetic interactions in the mutant, suggesting that MCFc, plays a role in plastid-mitochondrion communication. In silico analysis of MCFc implies a similar substrate-specific model to that of ADP/ATP carriers, although distinct motif differences in MCFc indicate potential variations in its function, with possible substrates including arginine, aspartate/glutamate, or citrate. These findings illuminate how mitochondrial energy contributes to fueling diatom photosynthesis.

ABSTRACT We recently described CRISPR/Cas9-based short homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans , a haploid budding yeast that is the most common cause of fungal meningitis and an emerging model organism. This was achieved by electroporation of strains stably expressing a codon-optimized Cas9 with two separate DNA molecules, one encoding a selectable marker flanked by short homology arms and a second encoding a sgRNA under the control of the U6 snRNA promoter. However, the efficiency of desired homology-dependent repair relative to undesired non-homologous end-joining (NHEJ) events can be low and variable. Here, we describe methods and strains enabling extremely efficient (∼99%) homology-dependent genome editing in C. neoformans . This high-efficiency method requires two manipulations. First, we placed the sgRNA-expressing segment into the marker-containing DNA flanked by targeting homology; thus, only a single DNA molecule is introduced into cells. Second, we used a strain mutant for the non-homologous end-joining factor Ku80 (encoded by YKU80 ). We also report the engineering of a yku80::amdS mutant strain harboring an insertion mutation that can be removed scarlessly via recombination between direct repeats. This enables the functional restoration of YKU80 after homology- dependent genome editing via selection against the amdS marker using fluoroacetamide. This approach minimizes documented drawbacks of using Ku-defective strains in downstream experiments. Finally, we describe a plasmid series that enables rapid cloning of sgRNA-marker constructs for genomic manipulation of C. neoformans , including gene deletion and C-terminal tagging. These methods, strains, and plasmids accelerate the genomic manipulation of C. neoformans .

Artificial intelligence (AI), including machine learning (ML) and deep learning (DL), has become an essential tool in modern agriculture, revolutionizing traditional practices and offering sustainable solutions to critical challenges, such as climate change, population growth, and resource scarcity. Through advanced algorithms and predictive models, ML and DL enhance precise genomic selection (GS), trait characterization, and the acceleration of crop breeding processes. These technologies facilitate the identification and optimization of key traits, including increased yield, improved quality, pest resistance, and tolerance to extreme climatic conditions. Additionally, ML-driven tools support gene-editing technologies, such as CRISPR-Cas9, contributing to the development of resilient and adaptable crops. By leveraging big data analytics and omic technologies, they provide valuable insights into linking genetic and phenotypic data, fostering the development of sustainable agricultural practices. This research explores the transformative potential of AI, particularly ML and DL, in Solanaceous crops by developing advanced breeding strategies to address challenges posed by climate change and rapid population growth. Furthermore, this study highlights the significant role of these technologies in creating novel crop varieties that are resilient to environmental stressors, while exhibiting superior agronomic and quality traits. AI and its applications, such as ML and DL, contribute to the genetic improvement of Solanaceous crops, strengthening agricultural resilience, ensuring food security, and promoting environmental sustainability.

Programmable CRISPR-Cas9 nucleases have become invaluable tools for genome editing. However, off-target cleavage by these nucleases could lead to unintended changes in the edited genome. Detection of off-target sites is critical to make genome editing technology safe and predictable. Although current in vitro methods for off-target detection can identify these sites, they are time-consuming, complex, and relatively costly. Here, we present CROFT-Seq ( CR ISPR nuclease of f- t arget detection by seq uencing), a sensitive, rapid, and cost-effective assay for the genome-wide detection of Cas9 off-target sites in vitro . CROFT-Seq performs comparably to the common currently used in vitro methods and serves as a valuable and efficient tool for the rapid assessment of genome-editing nuclease specificity. Notably, a high proportion of the top-ranked off-targets identified by CROFT-Seq were validated in cells, highlighting its effectiveness as a predictor of off-target sites.

Cisplatin chemotherapy of colorectal cancer (CRC) is associated with dose-limiting side effects and the development of drug resistance, resulting in reduced therapeutic effectiveness. The resistant phenotype in colon cancer is primarily due to changes in p53-regulated DNA damage signaling and /or defects in the cellular mismatch-repair pathway. Therefore, enhancing the efficacy of cisplatin chemotherapy remains a significant challenge. In this study, we used a TP53-KO patient-derived colon tumor organoid model to perform a genome-wide CRISPR KO screen in the absence and presence of cisplatin and identified gene knockouts that re-sensitize cisplatin-resistant TP53-KO colon cancer organoids to cisplatin treatment. Knockout of genes in the DNA Repair pathways, including Fanconi Anemia (FA cause re-sensitization of TP53-KO colon cancer cells to cisplatin. Inhibition of genes ERCC6, FANCL, and BRIP1 enhances cisplatin-induced cell death in TP53-KO colon cancer organoids. These findings suggest that targeting these pathways could be an effective approach to overcome chemoresistance of TP53-muatnt colon cancer cells to cisplatin.

Design: ing CRISPR single guide RNA (sgRNA) libraries targeting entire kingdoms of life will significantly advance genetic research in diverse and underexplored taxa. Current sgRNA design tools are often species-specific and fail to scale to large, phylogenetically diverse datasets, limiting their applicability to comparative genomics, evolutionary studies, and biotechnology. Here, we present ALLEGRO, a combinatorial optimization algorithm able to design minimal, yet highly effective sgRNA libraries targeting thousands of species. Leveraging integer linear programming, ALLEGRO identified compact sgRNA sets simultaneously targeting several genes of interest for over 2,000 species across the fungal kingdom. We experimentally validated the sgRNAs designed by ALLEGRO in Kluyveromyces marxianus, Komagataella phaffii , and Yarrowia lipolytica . In addition, we adopted a generalized Cas9-Ribonucleoprotein delivery system coupled with protoplast transformation to extend ALLEGRO’s sgRNA libraries to other untested fungal genomes, such as Rhodotorula araucariae . Our experimental results, along with cross-validation, show that ALLEGRO enables efficient CRISPR genome editing, supporting the development of universal sgRNA libraries applicable to entire taxonomic groups.

Abstract Bialleleic pathogenic variants in LCA5 cause one of the most severe forms of Leber congenital amaurosis, an early-onset retinal disease that results in severe visual impairment. Here, we report the use of gene editing to generate isogenic LCA5 knock-out (LCA5 KO) induced pluripotent stem cells (iPSC) and their differentiation to retinal organoids. The molecular and cellular phenotype of the LCA5 KO retinal organoids was studied in detail and compared to isogenic controls as well as patient-derived retinal organoids. The absence of LCA5 was confirmed in retinal organoids by immunohistochemistry and western blotting. There were no major changes in retinal organoid differentiation or ciliation, however, the localisation of CEP290 and IFT88 was significantly altered in LCA5 KO and patient photoreceptor cilia with extension along the axoneme. The LCA5-deficient organoids also had shorter outer segments and rhodopsin was mislocalised to the outer nuclear layer. We also identified transcriptomic and proteomic changes associated with the loss of LCA5. Importantly, treatment with the small molecules eupatilin, fasudil or a combination of both drugs reduced CEP290 and IFT88 accumulation along the cilia. The treatments also improved rhodopsin traffic to the outer segment and reduced mislocalisation of rhodopsin in the outer nuclear layer. The improvements in cilia-associated protein localisation and traffic were accompanied by significant changes in the transcriptome towards control gene expression levels in many of the differentially expressed genes. In summary, iPSC-derived retinal organoids are a powerful model for investigating the molecular and cellular changes associated with loss of LCA5 function and highlight the therapeutic potential of small molecules to treat retinal ciliopathies.

ABSTRACT A novel therapeutic strategy was recently proposed for high-risk neuroblastoma carrying copy number gain of the TRIM37 gene: centriole loss upon inhibition of polo-like kinase 4 (PLK4), while tolerated by normal cells, induces aberrant mitotic spindle formation and p53-dependent cell death in TRIM37 -overexpressing cells. Interestingly, while full PLK4 inhibition causes centriole loss, partial inhibition is known to elevate centriole numbers. Here we show using a novel selective PLK4 inhibitor RP-1664 that both centriole loss and amplification contribute to hypersensitivity of neuroblastoma cells. Whereas inactivation of TRIM37 and TP53 rescues neuroblastoma cell death at higher concentrations of RP-1664, at lower doses cell death is TRIM37/TP53 -independent. With CRISPR screens and live cell imaging we demonstrate that upon centriole amplification, neuroblastoma cells succumb to multipolar mitoses due to inability to cluster or inactivate supernumerary centrosomes. In vivo , RP-1664 shows robust efficacy in neuroblastoma xenografts at doses consistent with centriole amplification. STATEMENT OF SIGNIFICANCE High-risk neuroblastoma is associated with poor outcomes in pediatric patients and novel therapies need to be developed. We show that neuroblastoma cells are remarkably sensitive to PLK4 inhibitors due to a combination of two complementary mechanisms, supporting the evaluation of PLK4 inhibitors in clinical trials of high-risk neuroblastoma.

ABSTRACT In animals, collective cell migration is critical during development and adult life for repairing organs. It remains, however, poorly understood compared with single cell migration. Polymerization of branched actin by the RAC1-WAVE-Arp2/3 pathway is well established to power membrane protrusions at the front of migrating cells, but also to maintain cell junctions in epithelial monolayers. Here we performed a screen for novel regulators of collective cell migration by identifying genes associated with the RAC1-WAVE-Arp2/3 pathway at cell junctions using dependency maps and by inactivating these candidates using CRISPR/Cas9. In wound healing, MCF10A epithelial cells collectively migrate towards the free space in a coordinated manner. PKN2 knockout (KO) cells display decreased collective migration due to destabilization of adherens junctions, whereas MOB4 KO cells display increased collective migration with a swirling behavior. Upon wound healing, PKN2 relocalizes to lateral junctions and maintains coordinated migration in the monolayer, whereas MOB4 relocalizes to the front edge of cells migrating towards the wound. The role of MOB4 in controlling collective migration requires YAP1, since MOB4 KO cells fail to activate YAP1 and their phenotype is rescued by constitutively active YAP1. Together, our data reveal two complementary activities required for coordinating cells in collective migration.

ABSTRACT The construction of genome-wide DNA libraries from publicly available resources is essential for leveraging functional genomics to investigate complex biological systems. However, all existing high-throughput cloning methods for transferring DNA fragments between vectors require PCR amplification of the DNA fragments, rendering the construction of genome-wide DNA libraries labor-intensive and time-consuming. By introducing a concept of CRISPRshuttle cassette, we herein present a method named CRISPR-based shuttle cloning (CRISPRshuttle cloning). This method enables the high-throughput transfer of numerous DNA fragments from original plasmids with identical backbones to a different vector background without the need for PCR amplification of the DNA fragments. The procedure comprises two-step test tube reactions followed by bacterial transformation. Using CRISPRshuttle we successfully generated a library of GAL4/UAS-based UAS-ORF plasmids covering 1,397 human genes conserved in Drosophila . This library may serve as a valuable resource for gain-of-function screening in cultured cells and for the creation of a transgenic UAS-ORF library in Drosophila .

The complexity of molecular discovery requires autonomous systems that efficiently explore vast and uncharted chemical spaces. While integrating artificial intelligence (AI) with robotic automation has accelerated discovery, its application remains constrained in fields with scarce historical data. One such challenge is the design of lipid nanoparticles (LNPs) for mRNA delivery, which has relied on expert-driven design and is hindered by limited datasets. Here, we introduce LUMI-lab, a self-driving lab (SDL) system that enables efficient learning with minimal wet-lab data by integrating a molecular foundation model with an automated active-learning experimental workflow. Through ten iterative cycles, LUMI-lab synthesized and evaluated over 1,700 LNPs, identifying ionizable lipids with superior mRNA transfection potency in human bronchial cells compared to clinically approved benchmarks. Unexpectedly, it autonomously uncovered brominated lipid tails as a novel feature enhancing mRNA delivery. In vivo validation further confirmed that inhalation of LNPs containing the top-performing lipid, LUMI-6, achieved 20.3% gene editing efficacy in lung epithelial cells in murine models, surpassing the highest efficiency reported for inhaled LNP-mediated CRISPR-Cas9 delivery in mice to our knowledge. These findings demonstrate LUMI-lab as a powerful, data-efficient platform for advancing mRNA delivery, highlighting the potential of AI-driven autonomous systems to accelerate innovation in material science and therapeutic discovery.

Organisms that overwinter in temperate climates may experience freezing and freezing-induced oxidative stress during winter. While many insect species can survive freezing, molecular tools such as RNA interference (RNAi) or CRISPR have not been used to understand the physiological mechanisms underlying freeze tolerance. The spring field cricket Gryllus veletis can survive freezing following a 6-week fall-like acclimation. We used RNAi of an antioxidant enzyme in G. veletis to test the hypothesis that minimizing oxidative stress is important for freeze tolerance. In fat body tissue, Catalase mRNA abundance and enzyme activity increased during the acclimation that induces freeze tolerance. Other tissues such as midgut and Malpighian tubules had more stable or lower Catalase expression and activity during acclimation. In unacclimated (freeze-intolerant) crickets, RNA interference (RNAi) effectively knocked down production of the Catalase mRNA and protein in fat body and midgut, but not Malpighian tubules. In acclimated (freeze-tolerant) crickets, RNAi efficacy was temperature-dependent, functioning well at warm (c. 22°C) but not cool (15°C or lower) temperatures. This highlights a challenge of using RNAi in cold-acclimated organisms, as they may need to be warmed up for RNAi to work, potentially affecting their stress physiology. Knockdown of Catalase via RNAi in acclimated crickets also had no effect on the ability of the crickets to survive a mild freeze treatment, suggesting that Catalase may not be necessary for freeze tolerance. Our study is the first to demonstrate that RNAi is possible in a freeze-tolerant insect, but further research is needed to examine whether other genes and antioxidant molecules are important in freeze tolerance of G. veletis . Highlights Catalase expression and activity are elevated in freeze-tolerant cricket fat body RNAi knocks down Catalase in fat body and midgut at a warm temperature (22°C) RNAi is not effective at a cool temperature (15°C) that preserves freeze tolerance Catalase knockdown has no impact on survival of a mild freeze treatment The role of antioxidants in freeze tolerance warrants further study Graphical abstract

The impact of the genetic background on the lipidome of yeast strains remains underexplored. This study systematically compares the lipidomes of five commonly used laboratory yeast strains: BY4741, W303, D273-10B, RM11-1a, and CEN.PK2-1c. Shotgun lipidomics reveals significant variations in lipid class and acyl chain composition down to the level of molecular species. Notably, the most abundant lipid class differed between the strains: phosphatidylinositol (PI) lipids are predominant in BY4741, while phosphatidylethanolamine (PE) lipids are in D273. Ergosterol esters, which are the storage form of the major yeast sterol ergosterol, are at higher levels in all strains other than BY4741, correlating with a low gene expression of lipid metabolic enzymes Hmg1 and Are2 in BY4741. Despite these lipidomic differences, transcriptomic analysis did not show significant changes in most genes related to lipid metabolism, suggesting post-transcriptional modifications, protein abundance, and metabolic flux as potential regulatory mechanisms. This study underscores the complexity of lipidome regulation and the need for further investigation into the underlying mechanisms.

ABSTRACT Chromosomal deletion of tumor suppressor genes often occurs in an imprecise manner, leading to co-deletion of neighboring genes. This collateral damage can create novel dependencies specific to the co-deleted context. One notable example is the dependency on PRMT5 activity in tumors with MTAP deletion, which co-occurs with CDKN2A/B loss, leading to the development of MTA-cooperative PRMT5 inhibitors. To identify additional collateral damage context/target pairs for chromosome 9p and other common loci of chromosomal deletions, we conducted a combinatorial CRISPR screen knocking out frequently co-deleted genes in combination with a focused target library. We identified the gene encoding the ribosome rescue factor PELO as synthetic lethal with the SKI complex interacting exonuclease FOCAD, which is frequently co-deleted alongside MTAP and CDKN2A/B on chromosome 9p. A genome-wide screen in FOCAD isogenic cells further identified the ribosome rescue GTPase and PELO binding partner HBS1L as the top synthetic lethal target for FOCAD loss. Analysis of publicly available data and genetic manipulation of HBS1L using orthogonal modalities validated this interaction. HBS1L dependency in FOCAD -deleted cells was rescued by FOCAD re-expression, and FOCAD intact cells could be rendered HBS1L-dependent by FOCAD knockout, demonstrating the context specificity of this interaction. Mechanistically, HBS1L loss led to translational arrest and activated the unfolded protein response in FOCAD -deleted cells. In vivo , HBS1L deletion eliminated growth of FOCAD -deleted tumors. Here we propose a model where the FOCAD/SKI complex and HBS1L/PELO work together to resolve aberrant mRNA-induced ribosomal stalling, making the HBS1L/PELO complex an intriguing novel target for treating FOCAD -deleted tumors.

In eukaryotic cells, communication between organelles and the coordination of their activities depend on membrane contact sites (MCS). How MCS are regulated under the dynamic cellular environment remains poorly understood. Here, we investigate how Pex30, a membrane protein localized to the endoplasmic reticulum (ER), regulates multiple MCS in budding yeast. We show that Pex30 is critical for the integrity of ER MCS with peroxisomes and vacuoles. This requires the Dysferlin domain (DysF) on Pex30 cytosolic tail. This domain binds to phosphatidic acid (PA) both in vitro and in silico, and it is important for normal PA metabolism in vivo . The DysF domain is evolutionarily conserved and may play a general role in PA homeostasis across eukaryotes. We further show that ER-Vacuole MCS requires Pex30 C-terminal Domain of Unknown Function and that its activity is controlled by phosphorylation in response to metabolic cues. These findings provide new insights into the dynamic nature of MCS and their coordination with cellular metabolism.

Serine/Threonine phosphoprotein phosphatases (PPPs, PP1-PP7) are conserved metalloenzymes and central to intracellular signaling in eukaryotes, but the details of their regulation is poorly understood. To address this, we performed genome-wide CRISPR knockout and focused base editor screens in PPP perturbed conditions to establish a high-resolution functional map of PPP regulation that pinpoints novel regulatory mechanisms. Through this, we identify the orphan reductase CYB5R4 as an evolutionarily conserved activator of PP4 and PP6, but not the closely related PP2A. Heme binding is essential for CYB5R4 function and mechanistically involves the reduction of the metal ions in the active site. Importantly, CYB5R4-mediated activation of PP4 is critical for cell viability when cells are treated with DNA damage-inducing agents known to cause oxidative stress. The discovery of a dedicated PPP reductase points to shared regulatory principles with protein tyrosine phosphatases, where specific enzymes dictate activity by regulating the active site redox state. In sum, our work provides a resource for understanding PPP function and the regulation of intracellular signaling.

Telomere elongation is essential for the proliferation of cancer cells. Telomere length control is achieved by either the activation of the telomerase enzyme or the recombination-based Alternative Lengthening of Telomeres (ALT) pathway. ALT is active in about 10-15% of human cancers, but its molecular underpinnings remain poorly understood, preventing the discovery of potential novel therapeutic targets. Pooled CRISPR-based functional genomic screens enable the unbiased discovery of molecular factors involved in cancer biology. Recently, Optical Pooled Screens (OPS) have significantly extended the capabilities of pooled functional genomics screens to enable sensitive imaging-based readouts at the single cell level and large scale. To gain a better understanding of the ALT pathway, we developed a novel OPS assay that employs telomeric native DNA FISH (nFISH) as an optical quantitative readout to measure ALT activity. The assay uses standard OPS protocols for library preparation and sequencing. As a critical element, an optimized nFISH protocol is performed before in situ sequencing to maximize the assay performance. We show that the modified nFISH protocol faithfully detects changes in ALT activity upon CRISPR knock-out (KO) of the FANCM and BLM genes which were previously implicated in ALT. Overall, the OPS-nFISH assay is a reliable method that can provide deep insights into the ALT pathway in a high-throughput format.

ABSTRACT Machado-Joseph disease (MJD) is an autosomal dominantly-inherited neurodegenerative disorder, caused by an over-repetition of the polyglutamine-codifying region in the ATXN3 gene. Strategies based on the suppression of the deleterious gene products have demonstrated promising results in pre-clinical studies. Nonetheless, these strategies do not target the root cause of the disease. In order to prevent the downstream toxic pathways, our goal was to develop gene editing-based strategies to permanently inactivate the human ATXN3 gene. TALENs and CRISPR-Cas9 systems were designed to target exon 2 of this gene and functional characterization was performed in a human cell line. After the demonstration of TALEN’s and CRISPR-Cas9 efficiency on gene disruption, a sequence of each system was selected for further in vivo experiments. Although both TALENs and CRISPR-Cas9 systems led to a drastic reduction of ATXN3 aggregates in the striatum of a lentiviral-based mouse model of MJD/SCA3, only CRISPR-Cas9 system allowed the improvement of key neuropathological markers of the disease. Importantly, the administration of the engineered system in YAC-MJD84.2/84.2 mice mediated a delay in disease progression, when compared with non-treated littermates. These data provide the first in vivo evidence of the efficacy of a CRISPR-Cas9-based approach to permanently inactivate the ATXN3 gene in the brain of two mouse models of the disease, supporting its potential as a new therapeutic avenue in the context of MJD/SCA3.

ABSTRACT Pore-forming toxins (PFTs) are secreted bacterial effector molecules that disrupt host cell membranes. The α-hemolysin (HlyA) of uropathogenic Escherichia coli (UPEC) can exert damage to various mammalian cell types. While a candidate toxin receptor (CD11a/CD18 [LFA-1] integrin) exists on myeloid cells, the mechanism of HlyA cytotoxicity to epithelial cells remains undefined. We show that HlyA secretion by UPEC exacerbates renal tubular epithelial injury during ascending pyelonephritis in mice. A CRISPR-Cas9 loss-of-function screen in renal collecting duct cells identified clathrin-mediated endocytosis as required for HlyA cytotoxicity. HlyA internalization induces lysosomal permeabilization, facilitating protease release, cytoplasmic acidification, and mitochondrial dysfunction leading to rapid cell death. This mechanism contrasts with the described actions of other PFTs (plasma membrane poration and osmotic cytolysis). We also identify the low-density lipoprotein receptor (LDLR) as an epithelial receptor for HlyA; genetic or competitive inhibition of the HlyA-LDLR interaction prevented cytotoxicity. Our studies define a new mechanism of action for HlyA, in which its toxicity to epithelial cells requires LDLR-mediated, clathrin-dependent internalization. These results suggest therapeutic avenues for mitigating HlyA-induced damage during E. coli infections.

Streptococcus pneumoniae is an opportunistic pathogen responsible for life-threatening diseases including pneumonia and meningitis. The host defense against pneumococci relies heavily on macrophages, which can effectively internalize and degrade bacteria. Recent studies have implicated both canonical and non-canonical autophagy-related processes in bacterial clearance, but the precise pathways mediating defense against S. pneumoniae remain unknown. Here, we utilize a well-established zebrafish larval infection model to investigate the role of autophagy in host defense against pneumococci in vivo . Using a transgenic autophagy reporter line, we found the autophagy marker Lc3 being recruited to pneumococci-containing vesicles upon bacterial internalization by zebrafish macrophages. The genetic inhibition of core autophagy genes ( atg5 and atg16l1 ) led to loss of the Lc3 associations and their impaired acidification, significantly delaying bacterial clearance. This Lc3 recruitment is partially mediated by LC3-associated phagocytosis (LAP), as knockdown of cyba and rubcn moderately reduced Lc3 association with phagosomes and diminished pneumococcal degradation. Interestingly, we observed no involvement of xenophagy components in S. pneumoniae -infected macrophages, suggesting the activation of another non-canonical autophagy pathway, distinct from LAP, targeting pneumococci-containing phagosomes. Instead, we found that production of pneumococcal pore-forming toxin - pneumolysin induces LAP-independent Lc3 lipidation, which could be abolished by knockdown of tecpr1a indicating the involvement of the sphingomyelin-TECPR1-induced LC3 lipidation (STIL) pathway. Collectively, our observations shed new light on the host immune response against S. pneumoniae , demonstrating that two distinct non-canonical autophagy pathways mediate bacterial degradation by macrophages and providing potential targets for the development of novel therapies to combat pneumococcal infections.

Summary CHD3 is a component of the NuRD chromatin remodeling complex. Pathogenic CHD3 variants cause Snijders Blok-Campeau Syndrome, a neurodevelopmental disorder with variable features including developmental delays, intellectual disability, speech/language difficulties, and craniofacial anomalies. To unveil the role of CHD3 in craniofacial development, we differentiated CHD3 -KO induced pluripotent stem cells into cranial neural crest cells (CNCCs). CHD3 expression is low in wild-type iPSCs and neuroectoderm, but upregulated during CNCC specification, where it opens the chromatin at BMP-responsive enhancers, to allow binding of DLX5 and other factors. CHD3 loss leads to repression of BMP target genes and an imbalance between BMP and Wnt signalling, ultimately resulting in aberrant mesodermal fate. Consequently, CNCC specification fails, replaced by early-mesoderm identity, which can be partially rescued by titrating Wnt levels. Our findings highlight a novel role for CHD3 as a pivotal regulator of BMP signalling, essential for proper neural crest specification and craniofacial development.