Damage to the vascular endothelium is a major contributor to acute radiation injury in multiple organs that underlies acute radiation syndrome (ARS), yet there are no FDA-approved radiation countermeasures targeting endothelial cells. Use of kinome-scale CRISPR screens performed in cultured human vascular endothelial cells isolated from different organs identified CLK2 as a potential radioprotective target. Pharmacological inhibition of CLK2 using TG003 and Cirtuvivint protected these endothelial cells against radiation injury and reversed its effects across the transcriptome and phospho-proteome. Human Organ Chip models of human intestine and lung that contain organ-specific epithelium and microvascular endothelium faithfully replicated clinical features of ARS when exposed to radiation, which were prevented when treated with CLK2 inhibitors. Thus, CLK2 inhibitors may represent a new class of radiation countermeasure drugs that can protect multiple organs against radiation-induced toxicities in patients with ARS.

Apolipoprotein E ( APOE ) genotype is well known to influence both amyloid-β (Aβ) and tau pathologies and risk for Alzheimer’s disease (AD), but it also affects α-synuclein (α-syn) levels, Lewy pathology and risk of dementia in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). The APOE-R136S (Christchurch, CC) point mutation has been shown to protect against AD pathology and dementia, however, the molecular mechanisms underlying this protection and its effects on α-syn pathology are not well understood. Using CRISPR/Cas9 technology, we created a CC arginine-to-serine point mutation at the conserved location in mouse APOE (R128S) to understand its effects on Aβ, tau and α-syn pathologies. We crossed these APOE CC mice to 5xFAD, PS19 and A53T-αSyn-GFP (A53T) mice. Using these various double mutant mice, we tested the effect of mouse APOE CC on different proteinopathies, including Aβ, tau, Aβ-induced tau after paired helical filament (PHF)-tau intracortical injections, and α-syn after preformed fibril (PFF) intracortical and intramuscular injections. We used immunohistochemical, biochemical and behavioral measures to test for protective effects of APOE CC on these different proteinopathies. Heterozygous (Het) and homozygous (Hom) APOE CC mice showed increased plasma cholesterol and triglyceride levels, as seen in humans, but no differences in body or brain weight, or life expectancy. APOE CC decreased Aβ-induced tau pathologies in PHF-tau injected 5xFAD;Hom mice but did not change Aβ-plaque pathology in 5xFAD mice or tau pathology in PS19 mice. Although Aβ levels, tau levels and mouse sex correlated strongly with the behavioral performance, we only detected subtle effects of APOE CC on anxiety-like behaviors in crosses with 5xFAD, PS19 and PHF-tau injected 5xFAD mice. Interestingly, Het and Hom APOE CC mice both showed reduced formation and spread of Lewy pathology in brain after intracortical α-syn PFF injection and reduced formation in spinal cord after α-syn PFF injection into the hindlimb gastrocnemius muscle in A53T mice. Our study emphasizes the protective effects of the APOE CC variant against different proteinopathies important for dementia and movement disorders, including Aβ plaque, tau and α-syn, and suggests that targeting APOE CC could provide new therapeutic strategies for AD, DLB and PD. Graphical Abstract

Objective Primary liver cancer is a leading cause of cancer-related mortality and harbors recurrent mutations in chromatin regulators such as BRCA1-associated protein 1 (BAP1), yet their functional impact remains unclear. We investigated how BAP1 deficiency affects liver homeostasis and tumorigenesis to clarify its functional role. Design We employed inducible, liver-specific BAP1 knockdown in mice subjected to diet-induced metabolic stress (including rescue experiments), alongside autochthonous hydrodynamic CRISPR models, and profiled livers by RNA-seq, immunohistochemistry, and mass spectrometry-based lipidomics. Complementary mechanistic assays in liver cancer cells examined the unfolded protein response (UPR) under endoplasmic reticulum (ER) stress; findings were supported by immunohistochemical and transcriptomic analyses of BAP1-mutant patient samples. Results BAP1 safeguards liver homeostasis under diet-induced metabolic stress, as its loss triggers ER stress, hepatocyte death, and acute liver failure. Lipidomics revealed a shift toward ER-stress-associated dyslipidemia, and transcriptomics showed negative enrichment of fatty-acid metabolism and positive enrichment of UPR pathways. In contrast, BAP1 loss synergizes with oncogenic drivers to accelerate tumorigenesis in autochthonous liver cancer models, underscoring its context-dependent tumor suppressor function. Mechanistically, BAP1 directly regulates the ER stress mediator DDIT3 (CHOP) through chromatin remodeling, linking BAP1 loss to maladaptive stress responses. Consistently, elevated CHOP expression was observed in BAP1-mutant human liver cancers and other tumor types. Conclusion These findings establish BAP1 as a key chromatin regulator that connects stress adaptation to both liver homeostasis and tumorigenesis, highlighting the BAP1-UPR axis for future translational assessment. What is already known on this topic? BAP1 is a recurrently mutated chromatin regulator across cancers, including primary liver cancer, but its functional role in liver biology and tumorigenesis has remained unclear. Metabolic dysfunction-associated liver disease is a growing driver of liver tumorigenesis and is characterized by lipid imbalance, ER stress, and activation of the unfolded protein response. What this study adds? We show that BAP1 is a key chromatin regulator that integrates metabolic and ER stress responses in the liver. Loss of BAP1 undermines cellular adaptation to metabolic challenge and cooperates with oncogenic signals to promote liver tumorigenesis via dysregulated DDIT3 (CHOP) expression, linking chromatin control to hepatic stress resilience and disease progression. How this study might affect research, practice or policy? Our findings position the UPR-CHOP axis as a candidate therapeutic vulnerability in BAP1-deficient liver cancers, particularly in the context of MASLD/MASH, and provide a conceptual framework for targeting stress adaptation pathways in precision oncology.

One third of all emerging infectious diseases are vector-borne, with the vector’s ecology and physiology playing key roles in determining whether viruses can access new vertebrate host species and spread globally. Innate immunity is a known barrier to virus replication in mosquito vectors that influences arboviral vector tropism. We here generated novel CRISPR-Cas9-mediated knockouts of the NF-κB family transcription factor Rel2 in Aedes aegypti -derived Aag2 cells and tested the impact on the replication of a diverse range of arboviruses in the Flaviviridae and Togaviridae families and the class Bunyaviricetes . We found that NF-κB-mediated innate immunity has broad antiviral activity against the Ae. aegypti -borne orthoflaviviruses dengue virus (DENV), yellow fever virus (YFV) and Zika virus (ZIKV) in mosquito cells. In contrast, little impact of NF-κB-loss-of-function was observed for the alphavirus chikungunya virus (CHIKV) or phlebovirus Rift Valley fever virus (RVFV), indicating specificity in the antiviral effects of NF-κB-mediated immunity. By comparing orthoflaviviruses with different transmission routes (mosquito-borne, tick-borne, no known vector), we demonstrated that NF-κB-mediated immunity exerts its antiviral effects both early and late in the viral replication cycle, and that NF-κB-mediated immunity is not the only molecular barrier influencing the ability of orthoflaviviruses to replicate in Ae. aegypti cells. Overall, our work demonstrates the importance of mosquito NF-κB-mediated innate immunity in suppressing arbovirus replication, and shows that the barriers for arboviruses to adapt to new vector species are multifactorial and virus-specific. Our findings increase our understanding of the molecular barriers influencing arboviral emergence, and could inform the development of refractory mosquitoes incapable of transmitting human pathogens. Author Summary Mosquito-borne viruses are causing an increasing global disease burden due to urbanisation, globalisation, changing land use and the climate change-driven invasion of mosquitoes into new locations. Few vaccines and no medicines for mosquito-borne viral infections are licensed for human use. Historically, the most effective way to reduce human disease has been to kill mosquitoes using insecticides, which also harms beneficial insects and is leading to resistance. Newer technologies with lower ecological impacts are being developed, including genetically modified mosquitoes with a reduced ability to transmit viruses. Like humans, mosquitoes have an immune system that protects against viral infections, and strengthening this immune system through genetic modification has shown promise for reducing virus transmission. We here expand our understanding of the ways in which mosquito immunity could be harnessed by demonstrating that a gene called “Rel2” helps the mosquito fight mosquito-borne virus infection. Unlike previously studied mosquito immune responses, Rel2 has a very broad (but not universal) protective capacity against viruses. Our work also indicates that Rel2 works in concert with other arms of the mosquito immune system, highlighting a need for further research to fully realise the potential of genetically modifying mosquito immune responses for the prevention of human disease.

Cystic Fibrosis (CF) is a life-limiting genetic disorder caused by deleterious variants in the CFTR gene that results in altered mucous impairing the airway epithelia. Durable correction of these variants in airway cells remain a therapeutic challenge for ∼10% of individuals unresponsive to CFTR modulators. A common disease-causing CFTR splice site variant was corrected in primary CF airway cells using base editor RNAs. Single-cell RNA sequencing revealed a remarkable increase in detectable CFTR transcript in most CF airway epithelial cell types with notable enrichment of CFTR -expressing ionocytes and secretory goblet cells. Progenitor basal cell subtypes were edited but they decreased as a fraction of total cells and CFTR expressing cells compared to unedited cells. CRISPR base editors delivered by polymeric nanoparticles (PNPs) facilitated functional rescue of CFTR to clinically meaningful levels in immortalized and primary airway cells. PNPs delivered reporter encoding RNA to progenitor airway cells in fully differentiated airway cultures. Vitronectin was a major component of the PNP corona that formed in vivo, but pre-incubation with vitronectin did not enhance delivery. Together, these findings validate a scalable, non-viral platform with significant translational promise for treating CF and other respiratory diseases involving respiratory epithelial cell dysfunction.

ABSTRACT Colorectal cancer (CRC) cells are addicted to iron, which fuels nucleotide synthesis, mitochondrial respiration, and rapid proliferation. Yet paradoxically, high intracellular iron is cytotoxic to most other cells, raising the question of how CRC cells tolerate and exploit iron-rich environments. One pathway thought to mediate iron toxicity is ferroptosis, an iron-dependent form of cell death. However, most ferroptosis regulators were identified through synthetic chemical screens or small molecule activators, and it remains unclear whether these canonical pathways explain how iron itself triggers cell death, particularly in vivo . Here, using multi-omics profiling, CRISPR screening, and in vivo models, we uncover a heme–succinate dehydrogenase (SDH)–Coenzyme Q (CoQ) axis that enables CRC cells to buffer iron-induced oxidative stress. Heme-dependent SDH reduces CoQ, which redistributes to mitochondrial and plasma membranes to detoxify lipid ROS as a radical trapping antioxidant. This pathway functions alongside, and in some contexts independently of, canonical ferroptosis regulators. These findings reveal that CRCs co-opt metabolic cofactors not only for growth but also for survival under physiologically toxic iron levels, uncovering new vulnerabilities for therapy.

Conventional plant breeding relies on the occurrence of chromosomal crossover as well as spontaneous or non-targeted mutations in the genome induced by physical or chemical stressors. However, constraints exist concerning the number and variation of genotypes that can be achieved in this way as the occurrence and combination of mutations are not equally distributed across the genome. The underlying mechanisms and causes of reproductive constraints can be considered the result of evolution to maintain the genomic stability of a species, while at the same time allowing necessary adaptions. A continuous horizon scan was carried out to identify plants derived from new genomic techniques (NGTs) which show that CRISPR/Cas is able to circumvent at least some of these mechanisms and constraints. The reason for this is the specific mode of action: While physico-chemical mutagens such as radiation or chemicals merely cause a break of DNA, recombinant enzymatic mutagens (REMs) such as CRISPR/Cas additionally interfere with the cellular repair mechanisms. More recently developed REMs even expand the capabilities of NGTs to introduce new genetic variations within the target sequences. Thus, NGTs allow to introduce genetic changes and combinations that are unknown in the current breeding pool, and that are also unlikely to occur from any previously used breeding methods. The resulting genotypes may need to be considered as ‘new to the environment’. The reasons for the above can be identified in the mode of action of the REMs. CRISPR/Cas catalysed reactions in particular can interfere with and overcome 1) cytogenic features such as repair mechanisms; 2) factors influencing recombination and stability of the genome such as crossovers; 3) gene copies with and without proximity and 4) certain regulatory elements. The technical potential of NGTs should also be taken into account in regulatory provisions. Previously unknown genotypes and phenotypes may negatively impact plant health, ecosystems, biodiversity and plant breeding. It must further be acknowledged that the different outcomes of NGTs and conventional breeding are not always evident at first sight. As a starting point, within a process-oriented approval process, molecular characterisation can inform the following steps in risk assessment and guide requests for further data.

Aneuploidy is a hallmark of cancer and is a potential vulnerability that can be selectively targeted. To systematically identify genes that affect the incidence and fitness of aneuploid cells, we conducted a genome-wide CRISPR/Cas9 screen using NMS-P715, an inhibitor of the spindle assembly checkpoint (SAC) kinase Mps1/TTK. In this study, we identified a number of genes known to regulate aneuploidy and mitosis, and subsequently focused on PRR14L , a ubiquitously expressed gene previously implicated in chronic myelomonocytic leukemia (CMML). Proximity labeling of PRR14L using TurboID revealed several cell division proteins, including the PP2A-B56 phosphatase complex and the spindle assembly factor TACC3, as PRR14L-interacting proteins. Loss of PRR14L prolongs SAC-dependent mitotic arrest in response to microtubule depolymerization but, paradoxically, leads to catastrophic mitotic errors upon SAC abrogation by MPS1 inhibitors. A model derived from our findings provides a rationale for exploiting MPS1 inhibition as a potential vulnerability in cancers containing either PRR14L loss of function mutations or FGFR-TACC3 fusions.

The protective Icelandic mutation in the amyloid precursor protein (APP) gene, APP A673T , identified in Icelandic and other Nordic populations is associated with a significantly lower risk of developing Alzheimer’s disease (AD). Conflicting results have been reported for the APP A673T mutation in various knock-in models of AD, but its effect in 5x familial AD (5xFAD) mice has never been investigated. We have crossed C57Bl6/J mice expressing a single point mutation edited into the murine APP gene via CRISPR-Cas gene editing, termed APP A673T , with 5xFAD mice that overexpress human APP carrying the Swedish (K670N/M671L), Florida (I716V), and London (V717I) mutations as well as human presenilin-1 (PS1) with two mutations (M146L and L286V); the resulting mice were termed 5xFADxAPP A673T . We have investigated amyloid beta (Aβ) pathology in 5xFADxAPP A673T , 5xFAD and their respective controls, APP A673T and C57Bl6/J wild types, at 6-months of age using immunohistochemistry, immunoblotting, and ELISAs. We found a moderate yet significant reduction for Aβ plaque size in male 5xFADxAPP A673T compared to 5xFAD. No differences were observed for soluble/insoluble Aβ40 and Aβ42 levels per se, but lower plaque count/area was found in 5xFADxAPP A673T when Aβ42/Aβ40 ratios were low, suggesting a genotype-dependent sensitivity to Aβ aggregation and accumulation. Therefore, the APP A673T mutation has the potential to modify Aβ pathology in 5xFAD mice at the age of 6 months.

European chestnut is an agroforest species of great ecological, economic, and cultural importance in many temperate regions. However, in recent decades, it has been seri-ously threatened by various factors, including devastating diseases such as chestnut blight and ink disease, as well as the impacts of climate change. In this context, bio-technological tools have emerged as a key alternative for the protection, improvement, and sustainable use of the species. This paper analyzes the main biotechnological strategies applied to European chestnut. First, classical and assisted breeding tech-niques are discussed, including controlled hybridization and the use of molecular markers to accelerate the selection of genotypes of interest. In the field of molecular biotechnology, studies related to the identification of key genes, the development of genetic markers (SSR, SNP), and the omics characterization of chestnut are reviewed. The use of micropropagation techniques for the clonal multiplication of elite individu-als is also included. Furthermore, advances in genetic modifications are explored, highlighting the introduction of resistance genes through transgenic and cisgenic ap-proaches, as well as emerging technologies such as CRISPR/Cas9. Finally, future per-spectives for the application of biotechnology in the recovery, improvement, and sus-tainability of chestnut in the face of current and future threats are presented.

Dengue fever remains one of the most pressing vector-borne diseases worldwide, driven primarily by the mosquito Aedes aegypti. Recent advancements in biotech- nology—ranging from the use of Wolbachia-infected mosquito releases to genetic modification (GM) techniques such as RIDL and CRISPR-based suppression—have redefined vector control paradigms. This paper presents a comprehensive pro- tocol for the synthesis and evaluation of global biotechnological inter- ventions, designed to assess expectations, field efficacy, and ethical and ecological concerns. Drawing from both computational modeling and field trial literature, we integrate findings to inform an optimized vector management strategy for Jed- dah, Saudi Arabia. The strategy is structured according to the SPIRIT/TIDieR framework and supported by a simulation-based Design-of-Experiments (DoE) ap- proach. The protocol describes the interplay between biological interventions and stochastic modeling, emphasizing the need for localized parameter calibration, long-term monitoring, and participatory stakeholder engagement. This integrative review and modeling framework serves as a foundation for the rational design of large-scale, adaptive dengue suppression programs, demonstrating how simu- lated outcomes can guide real-world implementation.

Abstract Tomato brown rugose fruit virus (ToBRFV) has emerged as a significant threat to global tomato cultivation. While current diagnostic tools can detect ToBRFV, they are often expensive, technically demanding, and not easily adapted for use outside the laboratory. In this study, we developed a CRISPR-Cas12a trans-cleavage fluorescence assay integrated with PCR amplification for sensitive and specific detection of ToBRFV. The assay was developed using in silico-designed and chemically synthesised viral DNA templates, primers and CRISPR RNA (crRNA), enabling precise validation of Cas12a-mediated trans-cleavage activity. The use of a fluorophore-quencher (FQ) reporter allowed the direct visualisation of results under a portable blue/UV transilluminator. This PCR-Cas12a method demonstrated high sensitivity under the tested conditions and a faster turnaround, with visible detection possible in 30 min of Cas12a assay incubation following PCR amplification without the need for advanced equipment. This study highlights the advantages of Cas12a-based diagnostics and provides a foundation for developing rapid, efficient, and field-friendly assays for ToBRFV and other plant viruses.

Throughout several centuries, infectious pathogenic agents have been used as models for the ongoing efforts of vaccine development, which saved hundreds of millions of lives from life-threatening infectious diseases worldwide. Nonetheless, there has been a missing gap that various polymorphic microbes have been taking advantage of in their evolutionary pathway: the interferon system, which often prevented the timely activation of second and third-line host immunity, leading to chaotic and mismatching immune responses. The phenomenon of increased incubation period of various infectious diseases may be a result of the increased abilities of such microbial agents to directly and indirectly undergo molecular self-camouflaging, which prevents the activation of Type I and Type III Interferon-encoding genes (INGs) in indirect and direct manners respectively, and cleaves the mRNA molecules encoding such interferon glycoproteins, often causing major delays in the process of autocrine and paracrine signalling of Type I and Type III Interferon glycoproteins, which in turn allows an unrestricted, exponential increase of the microbial load/count, giving rise to a statistical probability that the quality of the delayed immune response will be low and contributory to the processes of pathogenesis and pathophysiology. Some microbial proteins as such also inhibit the translation of Interferon-Stimulated Genes, thereby substantially affecting the signalling rates within the cytokine system and often bringing a negative domino effect upon the activation rates of the adaptive immune system. Apprehending the foundational layer of the current problems in evolutionary microbiology, epidemiology and public health studies is most likely crucial for the course of immunological, pharmaceutical and vaccine-related clinical research. In the current case, it is the complex set of molecular capabilities to suppress Type I and Type III Interferon-based signalling displayed by several polymorphic microbes of public health concern, and it may be that the rates of immunopathogenesis induced by such microbes are directly proportional with such pathogenic abilities of induced interferon suppression. Proportional medical responses could include the development of approaches involving low dosages of human recombinant Type I and Type III Interferon glycoprotein and perhaps also of protollin in the nasopharyngeal cavity, potentially bringing an example of putting a novel concept of a “United Immune System” into practice. Furthermore, similar dosages of such interferons could be administered into human immune cells including plasmacytoid dendritic cells, as well as natural and adaptive lymphocytes, to optimise their immune function and integrity against various environmental hazards. Ultimately, clinical researchers may isolate the pathogenic agents, attenuate them through the process of loss-of-function laboratory research, before performing gene editing to insert Type I, Type III and perhaps also Type IV Interferon-encoding, perhaps as well as Pattern Recognition Receptor (PRR) Agonist-encoding genes that specifically match the PRR targeted by the implicated microbes, into their genomic profile and potentially releasing the genetically-modified pathogens back into the environment transmissible factories of Type I and Type III Interferons, perhaps as well as of specific PRR Agonist proteins, which could include outer membrane proteins from the B serogroup of Neisseria meningitidis bacteria. If the microbial genetic activities implicating evasion of the interferon system are too intense and multilateral, at least some of the microbial genes responsible for such activity could be permanently removed in some exchange with the human genes encoding major elements of the interferon system that would be inserted into the microbial genome afterward. It may be important to mention that the process of clinical weakening of the isolated microbes would be aimed at reducing the activity of microbial genes implicated in pathogenesis and pathophysiology, and perhaps not as much microbial genes involved in reproduction and transmission. Such a change may bring various pathogenic agents into a path of evolutionary self-destruction, as they would start producing and sending signals to the proximal, innate immune system as soon as they enter the first host cells, making their same processes of induced innate immune suppression ineffective, and several dilemmas in microbial evolution could ultimately be tackled as a result, possibly even at least attenuating the phenomenon of acquired antibiotic resistance by various pathogenic bacteria. A clinical approach as such is likely based on the model of increasing the accessibility to insulin-based treatment against Diabetes Mellitus via insulin-encoding gene insertion into the genomes of harmless bacteria prior to their administration into human host organisms, which saved millions of lives worldwide. Processes of shrinkage of any level of limitations to potential efficacy would include the manual utilisation of inhalators, oral drops and/or injectable serums containing such modified microbes to ensure that such an immunising effect would be conferred simultaneously with exposure to the artificially-changed genetic version of the microbe, effectively creating an “active evolutionary trap” for the pathogens, potentially resulting in their gradual de-selection whilst they continue to transmit just sufficiently enough to produce lasting immune memory. In other words, a phenomenon of “pathogen baptism” could occur, implicating a domination of “domestic variants” over wild-type variants in the environment, with the former becoming like “wild animals”, as they would remain the only virulent pathogenic variants and gradually even become extinct, with the “domestic” variants becoming dominant, according to the viral quasispecies theory. This set of clinical responses, including targeted immunoediting and gene vector strategies, can be analogized to a strategic operation against a mega-hurricane. The immune system, overwhelmed by storm-like chaos, cannot function effectively from the outside. Thus, medical intervention must act like military aircraft entering the eye of the storm from above—where calm resides—not to be engulfed, but to deploy stabilizing agents from within the calm zone. Only then can the storm’s structure be undone without triggering systemic devastation. This metaphor underscores the methodology of pathogen isolation, CRISPR-Cas9 attenuation, and IFN gene insertion, yielding feasible modifications with >85% editing efficiency and full cross-protection in preclinical models. A conquest from within, while remaining of another world. A set of clinical responses involving all such pathways may ultimately bring a promise of a health-related “Golden Age” throughout the world, with DeepSearch Artificial Intelligence (AI)-generated mathematical models indicating a significant probability that such a scenario would occur under real-world conditions - initially estimated at 60% via Grok 3 beta, refined to 62% via Grok 4 beta (November 2025), outperforming traditional mRNA vaccines (~39% prevention), whilst emphasising upon the high importance of the existence of thoroughly rigorous clinical testing steps and procedures to ensure no harm is caused in any such proposed candidate approaches, and to make sure that the world populations reach a full extent of informed consent.

CRISPR-based gene activation (CRISPRa) has emerged as a promising therapeutic approach for neurodevelopmental disorders (NDD) caused by haploinsufficiency. However, scaling this cis -regulatory therapy (CRT) paradigm requires pinpointing which candidate cis -regulatory elements (cCREs) are active in human neurons, and which can be targeted with CRISPRa to yield specific and therapeutic levels of target gene upregulation. Here, we combine Massively Parallel Reporter Assays (MPRAs) and a multiplex single cell CRISPRa screen to discover functional human neural enhancers whose CRISPRa targeting yields specific upregulation of NDD risk genes. First, we tested 5,425 candidate neuronal enhancers with MPRA, identifying 2,422 that are active in human neurons. Selected cCREs also displayed specific, autonomous in vivo activity in the developing mouse central nervous system. Next, we applied multiplex single-cell CRISPRa screening with 15,643 gRNAs to test all MPRA-prioritized cCREs and 761 promoters of NDD genes in their endogenous genomic contexts. We identified hundreds of promoter- and enhancer-targeting CRISPRa gRNAs that upregulated 200 of the 337 NDD genes in human neurons, including 91 novel enhancer-gene pairs. Finally, we confirmed that several of the CRISPRa gRNAs identified here demonstrated selective and therapeutically relevant upregulation of SCN2A, CHD8, CTNND2 and TCF4 when delivered virally to patient cell lines, human cerebral organoids, and a humanized mouse model of hTcf4 . Our results provide a comprehensive resource of active, target-linked human neural enhancers for NDD genes and corresponding gRNA reagents for CRT development. More broadly, this work advances understanding of neural gene regulation and establishes a generalizable strategy for discovering CRT gRNA candidates across cell types and haploinsufficient disorders.

Performing live-cell microscopy and high-dimensional gene expression measurements on the same cells is crucial for unraveling the molecular mechanisms underlying complex temporal phenotypes, yet this remains challenging using traditional approaches. To address these limitations, we developed Video Imaging with Spatial-Temporal Analysis by FISH (VISTA-FISH), a technique that matches live-cell recordings of cultured cells with the expression of thousands of genes in the same cell at the end of the video. Moreover, VISTA-FISH can simultaneously detect CRISPR guide RNAs in pooled screens, enabling investigation into the fundamental underpinnings of cellular activity, organelle transport, and other pivotal cellular functions. Using VISTA-FISH, we measured gene expression and neuron activity in the same differentiating neurons. This combined single-cell transcriptomic and video data allowed us to link differentiation stage, subcellular transcript localization, and cell subtype with neuron activity. Using these measurements, we built a model to predict activity from expression. Finally, we performed a pooled CRISPR interference screen with live-cell lysosome imaging, identifying alterations in lysosome movement and morphology as well as gene expression in perturbed neurons. Collectively, these studies position VISTA-FISH as a powerful new tool for elucidating the molecular mechanisms underlying neuron activity, organelle trafficking, and other complex temporal phenotypes.

ABSTRACT Circadian clocks orchestrate ∼24-hour cycles in gene expression, behavior, and physiology across most organisms 1 . Our recent study has revealed a striking spatial organization in the nucleus during the repression phase: core clock proteins such as Drosophila PERIOD (PER) are organized into distinct nuclear foci close to the inner nuclear envelope of clock neurons, and clock-regulated genes are similarly positioned in the nucleus 2 . However, the functional relevance of this subnuclear organization is unknown. Here, we investigate how the spatial organization of clock proteins and chromatin regulates circadian gene repression. Given that PER partners with TIMELESS (TIM) to enact transcriptional repression, we first investigated whether TIM is also a component of these nuclear foci. Using CRISPR-Cas9 to endogenously tag TIM with mNeonGreen, we performed high-resolution live imaging in Drosophila clock neurons. We found that TIM forms nuclear foci during the repression phase that co-localize with PER condensates, whereas TIM remains diffuse in the cytoplasm of per 01 null mutants. To probe the spatial relationship between these condensates and clock-regulated genes, we combined protein imaging with fluorescence in situ hybridization (FISH) which revealed that PER/TIM condensates were positioned adjacent to, but did not overlap with, clock gene loci during the repression phase. These results suggest that core clock proteins are spatially sequestered into repressive condensates away from chromatin, providing a new framework for understanding how nuclear architecture and phase separation together orchestrate rhythmic gene expression.

Cathepsins are papain-family cysteine proteases known to play a cell-intrinsic role in protein degradation in the lysosome, as well as in digesting ECM and surface proteins after being secreted. Both of these functions are known to mediate pro-tumorigenic effects of CTSB in a range of cancers. Here, we specifically investigate the role of CTSB in TNBC, an aggressive subtype of breast cancer, where we find that high expression of CTSB in TNBC is associated with better outcomes. We used CRISPR to knockout CTSB in two highly metastatic TNBC cell lines, MDA-MB-231 and MDA-MB-468, and find different effects. In MDA-MB-231 cells, knockout of CTSB has no effect on cell viability, increases tumor cell 3D invasion in an ECM-independent manner, and increases sensitivity to many standard of care chemotherapy drugs. However, in MDA-MB-468 cells, knockout of CTSB increases cell viability, decreases tumor cell 3D invasion, in an ECM-independent manner, and drives resistance to certain chemotherapy drugs without affecting response to others. We find that in these cells, CTSB is not secreted, and that differential downstream mTOR and Akt activation can explain the differences seen in these phenotypes. Overall, our studies demonstrate that CTSB can regulate TNBC cell phenotypes via its lysosomal cell-intrinsic role, but that effects are cell-line specific, suggesting potential heterogeneity in the role of CTSB in TNBC.

Inherited retinal degenerations (IRDs), driven by pathogenic mutations, often involve primary dysfunction of the retinal pigment epithelium (RPE) — a pathogenic feature shared with atrophic age-related macular degeneration (aAMD), despite aAMD’s multifactorial etiology. Prominin-1 (Prom1), traditionally linked to photoreceptor pathology, has an unclear role in RPE homeostasis. We assessed Prom1 expression in C57BL/6J mouse retina sections and RPE flat mounts using immunohistochemistry and generated Prom1-knockout (KO) mouse RPE cells via CRISPR/Cas9. Bulk RNA sequencing with DESeq2 and gene set enrichment analysis (GSEA) revealed Prom1-regulated pathways. Prom1-KO cells exhibited upregulation of Grem1, Slc7a11, Serpine2, Il1r1, and IL33, and downregulation of Ablim1, Cldn2, IGFBP-2, BMP3, and OGN. Hallmark pathway interrogation identified reduced expression of PINK1 (mitophagy) and MerTK (phagocytosis), implicating defects in mitochondrial quality control and outer segment clearance. Enrichment analysis indicated activation of E2F/MYC targets, mTORC1 signaling, oxidative phosphorylation, and TNFα/NF-κB signaling, alongside suppression of apical junction, bile acid metabolism, and EMT pathways. These findings suggest Prom1 safeguards RPE integrity by modulating stress responses, mitochondrial turnover, phagocytosis, metabolism, and junctional stability. Our study uncovers Prom1-dependent signaling networks, providing mechanistic insights into RPE degeneration relevant to both IRD and aAMD, and highlights potential therapeutic targets for preserving retinal health.

For viruses that replicate in the proximity of or bud at the endoplasmic reticulum (ER) associated membranes, proper processing of their glycoproteins is critical for successful infection. Rotavirus outer capsid protein VP7 is an ER-resident protein. However, its N-terminal signal peptide is removed by an unknown proteolytic mechanism. In this study, we leveraged tandem affinity purification followed by high-resolution mass spectrometry to profile host proteins that interact with VP7. We identified members of the signal peptidase complex (SPC) family as important host factors that facilitate rotavirus infection. CRISPR knockout or siRNA knockdown of distinct SPC subunits resulted in significant decrease in infectious rotavirus titers in a viral strain- and cell type-independent manner. While viral transcription, translation, and replication were not altered in the absence of SPC, we observed formation of abnormal viral particles by transmission electron microscopy (TEM) in SPCS1 knockout cells. Mechanistically, loss of SPC proteins led to inefficient cleavage of VP7 signal peptide and severely impaired the final steps of virion maturation and assembly. Additionally, we identified residue E256 within VP7 as a key site for SPC binding. An E to R mutation abolished VP7 interaction with SPC and subsequently led to reduced viral infectivity. Taken together, these findings define SPC as a novel regulator of VP7 maturation and rotavirus assembly and highlight its role as a novel cellular target for potentially broad-spectrum antiviral therapeutic development. Author summary Previous reports demonstrated that SPC is involved in the replication cycles of several members of the flavivirus family and human T-cell leukemia virus type 1 (HTLV-1). As an ER-resident outer capsid protein, rotavirus VP7 must undergo proper post-translational modifications, including the cleavage of its N-terminal signal peptide, to be functionally incorporated into mature virions. However, the processing mechanism remains unknown. For the first time, our study identifies SPC as an essential regulator of rotavirus assembly by mediating the cleavage of the VP7 signal peptide. Loss of SPC impairs VP7 signal peptide cleavage and maturation, thereby disrupting correct virion assembly and reducing infectious particle production. Using Alphafold3, we predicted the VP7 residue E256 to be at the binding interface with SPC complex. Experimentally, mutation of glutamic acid to arginine (E256R) substantially weakens this interaction and results in reduced viral propagation. Our findings unveil a novel post-translational checkpoint in rotavirus regulated by SPC and underscore the promise of SPC as a broad-spectrum antiviral target, especially for rotavirus, flavivirus, and HTLV-1, whose viral glycoproteins and structural proteins require SPC processing for proper maturation.

Abstract Xylella fastidiosa is a xylem-limited, vector-borne bacterial pathogen that infects a wide range of plant species worldwide, causing severe diseases and substantial economic losses in key crops such as olives, grapes, plums, almonds, and citrus. With few resistant cultivars available, current management strategies rely heavily on chemical control of insect vectors. Here, we report the isolation and characterization of a virulent bacteriophage, Savoy, exhibiting strong lytic activity against the grapevine-infecting X. fastidiosa strain WM1. Transmission electron microscopy identified Savoy as a Siphophage with a long contractile tail. The phage displayed optimal infectivity at a multiplicity of infection of 1 and remained stable across broad temperature (4–45 °C) and pH (3–11) ranges. In vitro assays demonstrated that Savoy effectively lysed planktonic bacterial cells and inhibited biofilm formation. Genome analysis revealed a 61,990 bp genome organized into functional modules associated with DNA metabolism, head and packaging, tail structure, lysis, and connector proteins. No known virulence factors, antimicrobial resistance genes, or CRISPR elements were detected. Multiple genes in Pseudomonas soli Savoy-resistant mutants harbored single nucleotide polymorphisms affecting diverse functional proteins. These findings highlight phage Savoy as a promising candidate for the development of sustainable phage-based biocontrol strategies against X. fastidiosa, with potential to reduce reliance on chemical treatments and mitigate crop losses.

Glucosylceramide synthase (GCS) catalyzes ceramide glycosylation in response to cell stress that produces glucosylceramide and other glycosphingolipids. GCS overexpression is a cause of drug resistance and enriches cancer stem cells (CSCs) during cancer chemotherapy. Previous studies showed that GCS modulates expression of p53 mutants and oncogenic gain-of-function (GOF) in heterozygous knock-in cell models ( TP53 R273H -/+ ). However, it is unclear whether GCS can modulate the effects of homozygous p53 mutations, which are common in many cancer cases. We report herewith that inhibition of GCS, via UGCG-knockout and using new inhibitor (Genz-161), effectively re-sensitizes drug resistance and diminishes CSCs in colon cancer cells carrying the homozygous p53 R273H mutation. In aggressive WiDr cells carrying TP53 R273H mutation, knockout of UGCG gene using CRISPR/Cas9 editing or inhibition of GCS with Genz-161 sensitized cancer cells to oxaliplatin, irinotecan and paclitaxel. With decreased ceramide glycosylation in lipidomic profiling, both UGCG-knockout and Genz-161 treatments substantially decreased wound healing, and diminished CSCs and tumor growth under chemotherapy. Interestingly, inhibition of RNA m 6 A methylation by neplanocin A reactivated p53 function and reversed drug resistance. Mechanistic investigation revealed that GCS inhibition downregulated METTL3 expression and repressed RNA-m 6 A modification on mutant p53 R273H effects. Altogether, our findings demonstrate that ceramide glycosylation promotes METTL3 expression and RNA m 6 A methylation in response to drug-induced stress, thereby promoting mutant p53 expression and associated GOF. Conversely, inhibition of GCS can diminish CSCs and drug resistance via reduction of m 6 A modification and reactivation of p53 function. GCS inhibition is an achievable approach for mutant cancer treatment.

Background BRCA1-associated protein 1 (BAP1) is a deubiquitinase, frequently altered in cancers including hepatocellular carcinoma and cholangiocarcinoma. While Bap1 has been shown to play key roles in metabolism, maintenance of tissue homeostasis, and immune cell development, little is known about its normal functions in the liver in vivo. This study aims to identify Bap1 specific effects on the liver’s immune microenvironment and biological functions. Methods Using AAV8-mediated CRISPR/CAS9 genome editing we generated a mouse hepatocyte-specific model of Bap1 knockout to define the changes that occur in liver biology in an in vivo system and characterize how loss of Bap1 alters the liver’s response to injury. Single-cell resolution spatial transcriptomics were performed in conjunction with immunohistochemistry to analyze cell-type composition and immune cell recruitment changes. Bulk RNA-sequencing was performed for further assessment of the impact of Bap1 loss on transcription. Results Hepatocyte-specific depletion of Bap1 induced transcriptional changes shared with acute injury. We observed a strong dysregulation of inflammatory pathways associated with BAP1 loss. Moreover, the transcriptional response of Bap1 depletion in hepatocytes to damage was markedly different than in control liver, with Bap1-deleted livers showing a decreased hepatocyte identity based on gene expression. Spatial transcriptomics and quantitative texture analysis of immunohistochemistry revealed an altered immune environment prior to damage and an impaired recruitment of immune cells in Bap1 depleted livers after damage. Conclusions Using a hepatocyte-specific Bap1 deletion we identified Bap1 as a critical modulator in the liver’s immune cell response. We show that Bap1 loss leads to an inflammatory environment prior to damage and disrupts the recruitment immune cells. Our quantitative spatial analysis highlights the power of such approaches to characterize the spatial distribution of different cell types in a tissue.

Malformations of cortical development (MCD) caused by variants in mTOR pathway genes (MPGs) are a leading cause of drug-resistant epilepsy. Characteristic histopathological features of MPG-associated MCD include cytomegaly and cortical dyslamination often with neurons in abnormally close apposition (aggregates). We hypothesized that cellular aggregation is an mTOR-dependent phenotype. Tsc2, Nprl3, Stradα , or Kptn were knocked out (KO) using CRISPR/Cas9 in N2a cells in vitro . Levels of phosphorylated ribosomal S6 protein (PS6; Ser240/244), a marker for mTOR activation, were defined via Western blotting in vitro . Timelapse live-cell imaging was used to observe aggregate formation, with or without mTORC1 inhibition (rapamycin). EdU-base cell proliferation assay and cell death assays were performed to determine whether aggregation was the result of changes in cell cycle or increased cell death. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to define changes in the cell aggregate proteome. Human MCD brain tissue specimens were stained with PS6 to assay mTOR signaling in neuronal clusters. All knockout lines formed multi-cell aggregates compared to control lines within 24-48 hours of plating in vitro . Aggregation was abolished with mTOR inhibitor treatment, establishing the mTOR-dependency of aggregate formation. Aggregation was not driven by cell proliferation, apoptosis/necrosis, or the presence of extracellular DNA in culture media. LC-MS/MS analysis revealed altered expression of protein across KO lines including adhesion molecules (e.g., contactin-3), cytoskeletal proteins (e.g., stathmin-2), and protein processing/transport (e.g., Uevld). Our findings establish aberrant cellular aggregation as an mTOR-dependent phenotype across multiple MPG associated with MCD. Changes in expression of adhesion molecules may contribute to abnormal cell aggregation and cortical lamination in MCD and results in abnormal network formation that leads to seizures. Highlights In human MCD specimens, neurons are frequently observed in clustered groups. In vitro models of mTORopathies show mTOR-dependent changes in cellular aggregation. Proteomic analysis revealed changes in protein levels in adhesion molecules and other molecules relevant to cellular dynamics and protein transport.

Congenital ectopia lentis (EL) poses a significant threat to visual outcomes in children. Although emerging causative genes have been identified, their biological functions remain poorly understood. In this study, we investigated the contribution of ADAMTSL4 variants to congenital EL in a cohort of 702 pedigrees. Pathogenic variants in ADAMTSL4 were identified in 23 probands (3.28%), comprising 29 distinct variants, with 14 being novel, making ADAMTSL4 the second most frequently mutated gene in the cohort. All affected individuals exhibited EL, and 26.09% also presented with ectopia pupillae (EP), also referred to as ectopia lentis et pupillae. To explore the functional consequences of ADAMTSL4 deficiency, we generated a CRISPR/Cas9-mediated adamtsl4 -knockout zebrafish model, that faithfully recapitulated cardinal human disease features with an incidence comparable to that observed in affected patients. Histological and ultrastructural analysis revealed disrupted zonular fiber anchorage at the lens capsule, even in mutated eyes without overt EL or EP. Transgenic overexpression of adamtsl4 successfully reversed the ocular phenotypes, confirming the gene’s essential role in ocular development. Single-cell RNA-sequencing and fluorescence in situ hybridization demonstrated enriched ADAMTSL4/adamtsl4 expression in the equatorial lens epithelium, retinal pigment epithelium (RPE), iris anterior pigmented epithelium, and choroid fibroblast. Functional assays using zebrafish and human RPEs revealed that ADAMTSL4 deficiency compromised cell adhesion and promoted cell migration. Transcriptomic profiling revealed significant enrichment of extracellular matrix organization and cell adhesion pathways, with cross-species validation identifying consistent upregulation of COL8A1/col8a1b . Notably, COL8A1 knockdown in ADAMTSL4 -deficient RPEs partially reversed the aberrant migratory phenotype, suggesting a functional interaction. Together, these findings establish ADAMTSL4 as a major causative gene in ectopia lentis et pupillae, highlight its role in orchestrating ocular integrity via regulation of extracellular matrix and cell behavior.

ABSTRACT Effective control of pest populations remains a major challenge for agriculture, public health, and conservation. While genetic control strategies such as the release of sterile males or individuals carrying dominant lethal alleles have achieved some success, they typically require repeated, large-scale releases due to immediate selection against costly alleles, limiting scalability and applicability to species that are difficult to rear in the laboratory. Recent CRISPR/Cas-based genome editing has enabled the development of more efficient genetic control methods that bias their own inheritance and impose a genetic load to achieve population suppression. These designs can potentially spread throughout an entire species range, making them unsuitable when only local or temporary suppression is needed. Here we explore self-limiting genetic strategies based on releasing males carrying autosomal genomic editors that create deleterious edits in essential genes. Unlike traditional approaches, where costly alleles are immediately purged, these editors can persist through multiple generations when creating female-specific or recessive edits, or when editing rates are less than 100%. This allows editors to survive in males, heterozygous carriers, or individuals unaffected by incomplete editing, trading immediate lethality for accumulated genetic load over time. Using deterministic population modelling, we demonstrate that homozygous releases of editors creating female-specific dominant or recessive edits require 45% and 50% fewer males than releasing individuals carrying dominant lethal alleles (RIDL), respectively, to achieve comparable suppression levels. Efficiency gains are further enhanced by targeting multiple genes simultaneously, with editors making female-specific recessive edits showing approximately 50% reduction in release requirements when targeting three genes compared to one – representing a 73% reduction compared to RIDL. Co-releasing editors with a second construct that temporarily boosts editor frequency can achieve efficiency comparable to previously proposed selectively neutral designs while maintaining temporal self-limitation. These results highlight promising alternative strategies for achieving localised, efficient, and self-limiting pest population control suitable for contexts requiring contained suppression.