Anoikis is an apoptotic cell death program triggered upon detachment from surrounding extracellular structures. However, the ability to evade cell death by anoikis in the presence of apoptosis-inducing stimuli is necessary for the formation of malignant tumors and progression to metastasis. Our findings indicate that the BRN2 (POU3F2) transcription factor is associated with anoikis resistance in melanoma cells. However, the BRN2 signaling cascade driving anoikis resistance remains unknown. Herein, we employed genome-wide CRISPR screens to validate BRN2 as a driver of anoikis resistance. Small molecule inhibition of BRN2 in melanoma cell lines with acquired anoikis resistance resensitized to death by anoikis in ultra-low attachment conditions. Our quantitative mass spectrometry analysis revealed that BRN2 functionally impacts oxidative phosphorylation and mitochondrial activity, whereby probes designed to inhibit BRN2 induced apoptosis and mitochondrial fragmentation through the MAPK and NF-κB signaling pathways and reduction in PPARɣ expression. Our study suggests that inhibition of BRN2 might allow the targeting of metastatic cells in circulation, and sensitizes cells to BRAF-targeted therapy, improving the prognosis for melanoma patients. Abstract Figure Graphical Abstract. Role of BRN2 in driving anoikis resistance in melanoma. Upon detachment from the extra-cellular matrix (ECM) melanoma cells must evade cell death by anoikis to seed distant metastases. This study expanded the understanding of the role of the BRN2 transcription factor as a driver of resistance to anoikis in melanoma. The use of small molecule inhibitors targeting BRN2 revealed that the transcription factor drives anoikis resistance via the MAPK and NF-κB signaling pathways, resulting in PPARγ dysregulation and subsequently driving mitochondrial dysfunction. Green boxes = previously published drivers of anoikis resistance in melanoma. Blue box = changes to mitochondrial function following inhibition of BRN2 as determined by proteomics analysis.

CRISPR/Cas9-based homing gene-drives (homing-drives) hold enormous potential as control tools for mosquito disease-vectors. These genomically-encoded technologies spread themselves through target populations by creating double-stranded DNA breaks on homologous chromosomes, into which the homing-drives are copied (‘homed’). Homing is dependent on sequence homology between the genomic regions flanking the transgene insertion and the break site. Homing efficiency (i.e. copying rate) substantially impacts the power of these systems: less efficient homing-drives spread slower, have fewer applications and are more resistance-prone. Understanding what influences homing-drive efficiency is therefore vital to the successful use of these technologies. Here we report a novel mechanism by which a homing-drive’s efficiency can be significantly impaired by natural sequence variation within a population into which it is spreading. Using a kmo -targeting ‘split’ homing-drive in the West Nile virus mosquito Culex quinquefasciatus , we found that target-site heterology (sequence mismatch between the genomic regions flanking the target cut-site and the homing-drive transgene) of less than 10% reduced homing efficiency by up to 54%. While substantial research effort has been dedicated to increasing homing-drive efficiency through optimisation of within-construct components, our results highlight that the real-world efficacy of these systems may in part depend on variation beyond these controllable factors.

Abstract Tn7 mobile genetic elements are known for their sophisticated target-site selection mechanisms and, in some cases, programmability. Recognition of target sites is mediated by designated transposon-encoded proteins and modulated by host factor proteins. In the case of the CRISPR-associated Tn7 elements from the type V-K, the ribosomal protein uS15 is an integral component of recruitment complex that promotes R-loop completion. Previous biochemical work also revealed that the ribosomal protein uL29 and the acyl carrier protein (ACP) influence Tn7 transposition frequency in vitro . However, how uL29 and ACP regulate the formation of the Tn7 targeting complex remains unclear. The prototypical Tn7 element encodes a heteromeric transposase (TnsAB), a AAA+ adaptor (TnsC), and two target-site selection proteins (TnsD and TnsE). TnsD targets a highly conserved site at the end of the glmS gene ( attTn7 ). However, poor protein stability has precluded the molecular characterization of how TnsD recognizes its target site. Here, we show that ACP and uL29 interact with the C-terminal region of TnsD through reciprocal electrostatic interactions, in turn, mitigating its tendency to aggregate. Additionally, we identify the uL29 and ACP residues that mediate the interaction with TnsD and stimulate DNA binding. These results unveil unique features of the TnsD-mediated target-site selection complex.

CRISPR/Cas9-based genome editing in the model bryophyte Physcomitrium patens (commonly known as Physcomitrella) is widely used for gene knockout via small insertions or deletions (indels). However, this approach may leave residual gene activity and typically requires sequencing-based validation. In this study, we established an efficient strategy for generating large, targeted deletions across multiple genes using dual-gRNA targeting. We first compared the efficiency of polycistronic tRNA-gRNA arrays to conventional gRNA constructs expressed under individual promoters, using the checkpoint protein gene MAD2 as a target. We found that a polycistronic construct doubled the frequency of large gene deletions compared to a conventional design. We then demonstrated that simultaneous deletion of two or four genes, targeting the katanin and TPX2 gene families, respectively, can be achieved in a single transformation event. The polycistronic system also increased deletion frequencies in the multiplex context, with up to 42% efficiency for individual genes and successful recovery of quadruple mutants. As a drawback, we confirmed that deletion efficiency varied substantially among individual gRNA pairs, indicating that gRNA design remains a critical factor in multiplex editing. This study establishes a versatile and scalable framework for generating multi-gene deletion mutants in P. patens , facilitating functional genomics and biotechnological applications requiring precise gene removal.

In contrast to animals, plants have a high regenerative capacity, and they can form new organs and even complete individuals from a few cells present in adult tissues, either in response to injury or to the alteration of their environment. In this study, we describe the isolation and characterization of the more adventitious roots1-1 ( mars1-1 ) mutant, which exhibits enhanced regenerative potential upon wounding in tomato hypocotyl explants. Additionally, the mars1-1 fruits exhibited a rough surface due to the ectopic proliferation of subepidermal cells, which formed callus-like structures on the cuticle. The MARS1/ROUGH gene encodes a conserved lysine-specific histone demethylase, SlLSD1, which regulates a variety of processes in metazoans, including cell proliferation, stem cell pluripotency, and embryogenesis. Two CRISPR/Cas9 null alleles, mars1-2 and mars1-3 , were generated and their pleiotropic phenotype was characterized. We found elevated levels of H3K4me1 in mars1/rough seedlings, which suggests that SlLSD1 is required for the demethylation of this histone mark. To ascertain the impact of altered epigenetic marks in the mars1/rough mutants on gene expression regulation, we conducted a transcriptome analysis using a variety of RNA-Seq studies on tomato hypocotyls. By employing specific bioinformatic workflows and leveraging on the resolution of directional RNA-Seq data, we have identified over several dozen distinct genomic regions that exhibit de novo expression in the mars1/rough mutants. One such region includes a novel B-type cyclin gene, which is upregulated in the mars1/rough mutants and may account for the observed phenotypes. Our findings indicate that SlLSD1 plays a role in the establishment and maintenance of silencing in specific genomic regions that are essential for tissue-specific reprogramming.

Adeno associated virus (AAV)-mediated delivery of CRISPR associated nucleases (AAV-CRISPR) is a promising solution to treat genetic diseases such as Duchenne Muscular Dystrophy (DMD) and is now in early clinical trials. However, genotoxicity and immunogenicity concerns have hindered clinical translation. Due to the complex etiology associated with DMD, the post-transduction consequences of double-stranded breaks induced by AAV-CRISPR in disease models are unclear. This barrier is partially conferred by conventional sequencing methods where common outcomes of AAV-CRISPR editing often escape detection. However, recent reports of novel long-read sequencing approaches permit comprehensive variant detection using a broader sequence context. Here, we comprehensively investigated genomic and transcriptomic post-AAV-CRISPR transduction consequences in myoblast cells and a DMD mouse model following intramuscular and intravenous AAV-CRISPR therapy using both long- and short-read sequencing techniques. Structural variant characterization indicates that unintended on-target large insertions and inversions are common editing outcomes. We demonstrate that combining adaptive sampling with nanopore Cas9-targeted sequencing (AS-nCATS) for long-read quantification of AAV integration is synergistic for detecting difficult-to-amplify editing events. This unbiased data suggests that full-length AAV integration is equally as probable as the on-target deletion. Further, we develop a Nanopore Rapid Amplification of cDNA Ends (nRACE-seq) pipeline for long-read detection of unknown 5’ or 3’ ends of edited transcripts. The nRACE-seq approach effectively detects the presence of AAV- Dmd chimeric transcripts, erroneous splicing events, and off-target AAV integration sites. In summary, our findings offer insights into the adaptation of AAV-CRISPR DSB-mediated therapeutics for monogenic diseases and promote the standardization of CRISPR evaluation. We highlight the importance of coupling polymerase-based and polymerase-free methods in long-read sequencing to assess editing outcomes as the field progresses toward clinical applications.

The transcription factor STAT3 plays broad roles in epithelial biology, yet its function in human esophageal development remains undefined. Using 2D and 3D human induced pluripotent stem cell (hiPSC)-derived platforms, we investigated how STAT3 regulates esophageal epithelial differentiation. We find that STAT3 is dispensable for definitive endoderm and anterior foregut endoderm specification but becomes essential during the transition to esophageal progenitor cells (EPCs). Inhibition of STAT3, via CRISPR-mediated knockout or siRNA, impairs the expression of key EPC and differentiation markers, including TP63 , and disrupts 3D organoid formation. These defects are accompanied by reduced epithelial proliferation. Notably, STAT3 is highly expressed in human fetal esophageal tissues and hiPSC-derived organoids, while its deletion in the developing mouse esophagus does not affect epithelial architecture, highlighting species-specific differences. Together, these findings identify STAT3 as a critical determinant of basal cell identity and epithelial morphogenesis, revealing a developmental checkpoint in early human esophageal lineage commitment.

The DNA-incorporating nucleoside analogs azacytidine (AZA) and decitabine (DEC) have clinical efficacy in blood cancers, yet the precise mechanism by which these agents kill cancer cells has remained unresolved -- specifically, whether their anti-tumor activity arises from conventional DNA damage or DNA hypomethylation via DNA methyltransferase 1 (DNMT1) inhibition. This incomplete mechanistic understanding has limited their broader therapeutic application, particularly in solid tumors, where early clinical trials showed limited efficacy. Here, through the assessment of drug sensitivity in over 600 human cancer models and comparison to a non-DNA-damaging DNMT1 inhibitor (GSK-3685032), we establish DNA hypomethylation, rather than DNA damage, as the primary killing mechanism of AZA and DEC across diverse cancer types. In further support of an epigenetic killing mechanism, CRISPR drug modifier screens identified a core set of chromatin regulators, most notably the histone deubiquitinase USP48, as AZA and DEC protective factors. We show that USP48 is recruited to newly hypomethylated CpG islands and deubiquitinates non-canonical histones, establishing USP48 as a key molecular link between the two components of epigenetic gene regulation: DNA methylation and chromatin modification. Furthermore, loss of USP48, which occurs naturally through biallelic deletions in human cancers, sensitized both hematologic and solid tumors to DNMT1 inhibition in vitro and in vivo. Our findings elucidate the epigenetic mechanism of action of AZA and DEC and identify a homeostatic link between DNA methylation and chromatin state, revealing new therapeutic opportunities for DNMT1 inhibitors in solid tumors.

Upstream open reading frames (uORFs) are short translated regions that occur in the 5□ untranslated regions (5□ UTRs) of mRNA transcripts where they primarily serve to repress expression translation of the downstream primary open reading frame (pORF). Their widespread presence across mammalian transcriptomes suggests an important role in shaping the proteome, although the mechanistic basis of their regulatory effects remain incompletely understood. Here we present an integrated experimental and computational investigation into the features that govern uORF-mediated translation control. Using high-resolution proteomics data from 29 healthy human tissues and machine learning-based simulations, we have systematically dissected how features including uORF length, amino acid composition, start codon position, stop codon position, and Kozak context influence repressive activity, and performed experimental validation using reporter gene constructs. We also investigated how multiple uORFs within a single 5□ UTR can interact in synergistic or antagonistic ways, with the potential to produce counterintuitive effects on pORF translation. From these studies, we present a model of uORF function, suggesting a hierarchy of uORF feature importance, and proposing that a combination of uORF translation initiation probability, ribosome recycling rate, intercistronic ternary complex recharging requirements, and ribosome stalling mechanisms underlie uORF repressive activity. Together, these studies provide a comprehensive view of the molecular logic underlying uORF activity, offering new insights into their endogenous and highlighting their potential as targets for drug development.

Zonula occludens-1 (ZO-1), encoded by the TJP1 gene, is a crucial scaffolding protein within tight junctions that maintains epithelial and endothelial barrier integrity. In addition to its structural role, ZO-1 participates in signal transduction pathways that influence various cellular processes such as proliferation, differentiation, and apoptosis. Increasing evidence suggests that tight junction proteins, including ZO-1, play important regulatory roles in tumor progression, particularly by modulating metastasis, cell polarity, and vascular remodeling. Ovarian cancer, the most lethal gynecologic malignancy, is characterized by rapid growth, peritoneal dissemination, and a strong reliance on tumor angiogenesis. However, the specific role of ZO-1 in regulating angiogenesis within ovarian cancer remains poorly defined. In this study, we used CRISPR-Cas9-mediated gene editing to generate TJP1 knockout (KO) ovarian cancer cell lines and investigated the impact of ZO-1 loss on the expression of angiogenesis-related genes. Transcriptomic and qRT-PCR analyses revealed upregulation of KLF5 and IL-8, both of which are well-established pro-angiogenic factors. Furthermore, functional assessment using a Matrigel™ tube formation assay demonstrated that conditioned media from ZO-1-deficient cells significantly enhanced endothelial tube formation. These findings indicate that ZO-1 loss promotes a pro-angiogenic tumor microenvironment, likely through modulation of key signaling molecules such as KLF5 and IL-8. Therefore, ZO-1 may serve as a potential suppressor of angiogenesis and a therapeutic target in ovarian cancer.

ABSTRACT Embryonic development is precisely shaped by maternal and zygotic factors. These maternal factors exert their influence through maternal effects, a phenomenon where an offspring’s phenotype is determined, at least in part, by the mother’s environment and genotype. While environmental maternal effects can cause phenotypes that present both early and later in life, genetic maternal effects generally induce phenotypes in the earliest embryonic stages. Here, we reveal a genetic maternal effect that influences the development of cells that arise after early embryogenesis, highlighting that specific cell types can be susceptible to late-onset genetic maternal effects. Using zebrafish to study microglia, the resident immune cells of the brain, we identified a mutation in sry-related HMG box gene-17 (sox17) that exhibits a maternal effect phenotype that presents as a reduction of microglia in the brain and precursors in the yolk sac. We demonstrate that sox17 is expressed in microglia and their yolk sac precursors and is maternally-loaded. We show that sox17 restoration via embryonic injection reverses the maternal effect on microglia and yolk sac cells in sox17 mutants. To identify additional genes interacting with sox17 , we nominated genes from scRNA sequencing analysis of mouse embryonic microglia to perform a genetic screen using CRISPR mutagenesis and a custom-built robot that captures confocal images of the zebrafish brain in high-throughput. This screen identified f11r.1, gas6, and mpp1 as modifiers of microglia abundance in the embryonic brain, which we demonstrated are also expressed in zebrafish microglia. Transcriptional and mutant analyses with these new modifiers suggest that sox17 positively regulates mpp1 transcription. These results demonstrate that microglia are susceptible to genetic maternal effects, in addition to their known sensitivity to environmental maternal effects. Our findings reveal a late-onset phenotype associated with the maternal genotype, expanding the recognized impact of genetic maternal effects beyond initial embryo viability and into long-term vigor.

Pathogenic KCNQ2 variants are associated with developmental and epileptic encephalopathy (KCNQ2-DEE), a devastating disorder characterized by neonatal-onset seizures and impaired neurodevelopment with no effective treatments. KCNQ2 encodes the voltage-gated potassium channel K V 7.2, which regulates action potential threshold and repolarization. However, the relationship between K V 7.2 dysfunction and abnormal neuronal activity remains unclear. Here, we use human induced pluripotent stem (iPSC)-derived neurons from 5 KCNQ2-DEE patients with pathogenic variants and CRISPR/Cas9-corrected isogenic controls to investigate pathophysiological mechanisms. We identify a common dyshomeostatic enhancement of Ca 2+ -activated small conductance potassium (SK) channels, which drives larger post-burst afterhyperpolarizations in KCNQ2-DEE neurons. Using microelectrode arrays (MEAs), we recorded over 18 million extracellular spikes from >8,000 neurons during 5 weeks in culture and then applied supervised and unsupervised machine learning algorithms to dissect time-dependent functional neuronal phenotypes that defined both patient-specific and shared firing features among KCNQ2-DEE patients. Our analysis identified irregular spike timing and enhanced bursting as functional biomarkers of KCNQ2-DEE and demonstrated the significant influence of genetic background on phenotypic diversity. Importantly, using unbiased machine learning models, we showed that chronic treatment with the K V 7 activator retigabine rescues the disease-associated functional phenotypes with variable efficacy. Our findings highlight SK channel upregulation as a critical pathophysiological mechanism underlying KCNQ2-DEE and provide a robust MEA-based machine learning platform useful for deciphering phenotypic diversity amongst patients, discovering functional disease biomarkers, and evaluating precision medicine interventions in personalized iPSC neuronal models.

TGF-β-mediated signaling controls mast cell (MC) development and exerts anti-inflammatory functions, while antigen/allergen (Ag)-triggered FcεRI activation commands pro-inflammatory reactions. TGF-β induces strong C-terminal and low linker phosphorylation of SMAD2. In contrast, Ag triggers immediate, MEK-dependent SMAD2 linker phosphorylation only. Both stimuli can positively or negatively influence each other’s effects on MC activation in a gene-dependent manner. However, the molecular and cellular mechanisms of SMAD2 in MCs still need to be elucidated. To decipher the role(s) of SMAD2 in MCs, SMAD2 was ablated in PMC-306 MCs using CRISPR/Cas9, and the effects were studied after TGF-β and/or Ag stimulation. The absence of SMAD2 led to increased proliferation and survival, as well as decreased transcription of target genes like Smad7 and Jun in steady state and after TGF-β treatment. Interestingly, SMAD2 was found to regulate the strength and kinetics of TGF-β-mediated SMAD1/5 activation, resulting in augmented expression of genes like Id2 and Id3 in SMAD2-deficient MCs. Unexpectedly, SMAD2 was observed to license Ag-triggered production of pro-inflammatory cytokines, such as IL-6 and TNF, by monitoring expression of secondary repressive signaling elements. Re-introducing SMAD2 restored these events with varying sensitivity depending on the receptor system triggered. Our findings reveal SMAD2 as an initial hub in TGF-β-SMAD1/5 and Ag-FcεRI signaling, offering new possibilities for therapeutic intervention in both TGF-β-controlled and Ag-triggered MC functions using potential SMAD2 activators or inhibitors.

Centriolar satellites (CS) are ubiquitous, membrane-less organelles recognized for organelle crosstalk, plasticity, diverse functions and links to developmental and neuronal diseases. However, the molecular principles governing their assembly and regulation remain poorly understood. To address this, we developed cellular and in vitro biogenesis assays that allow spatiotemporal quantification of CS granule properties during assembly, remodeling and maintenance. Using these tools, we show that CS assemble via a hierarchical pathway initiated by PCM1 scaffold formation followed by regulated client recruitment. PCM1 intrinsically assembles into granules through multimerization, a process modulated by cytoskeleton. High-resolution imaging revealed that PCM1 and its clients occupy distinct subdomains with different compositions and dynamics, adding an additional layer of regulation. Perturbing PCM1 multimerization impaired ciliary signaling, underscoring its functional importance. Collectively, these findings define the molecular basis of CS biogenesis, establish new tools to probe their context-dependent functions, and provide a framework for understanding how CS deregulation contributes to disease. More broadly, the principles uncovered here may extend to other membrane-less organelles, explaining their specificity and plasticity.

Trafficking from the endoplasmic reticulum to the Golgi apparatus comprises the first steps toward the correct localization of 30% of eukaryotic proteins. Coat protein complexes COPII and COPI are involved in forward and retrograde transport of cargo and cargo receptors between the ER and the Golgi. Although COPII forms coated vesicles in vitro, the biogenesis, morphology and organization of transport carriers in mammalian cells is debated. We use in situ cryo-electron tomography and super-resolution fluorescence microscopy to reveal the molecular architecture of ER exit sites in human cells. We visualise ribosome-exclusion zones enriched with COPII and COPI-coated vesicles and thus resolve the debate regarding the existence of COPII coated vesicles. COPII vesicles derive from ER membranes, whereas COPI vesicles originate from the ER-Golgi intermediate compartment. We quantify coated vesicle morphology and positioning with respect to other ER exit site components, providing a molecular description of the mammalian early secretory pathway.

ABSTRACT CRISPR-Cas12a is a programmable, RNA-guided endonuclease that has revolutionized biotechnology, with applications in genome engineering and diagnostics. To induce nuclease activity, Cas12a must first interact with the target dsDNA duplex by associating with a short protospacer adjacent motif (PAM) in the sequence. In this study we have split this target duplex to create PAM-proximal and PAM-distal duplex regions, which has allowed us to regulate trans-cleavage activity when these regions are included in combination or separately. These observations on Cas12a activity led to hypotheses into the related functional mechanisms, which we have tested and that have highlighted DNA/protein interactions during Cas12a complex assembly that were not otherwise apparent. Selective destabilization of the nucleic acid complexes appears to drive greater reliance on the Cas12a protein for complex stability. We have exploited this to provide significant improvements in both structural selectivity and nucleotide specificity in PAM-proximal and PAM-distal duplex regions, respectively. The result is an architecture that shows promise as a PAM-free ultra-specific platform to resolve single nucleotide polymorphisms. GRAPHICAL ABSTRACT

Shikonin, a 1,4-naphthoquinone derivative produced by several Boraginaceae species, exhibits unique pharmacological properties and is used as a natural dye. The regulatory factors of shikonin production have been demonstrated using a cell culture system of Lithospermum erythrorhizon . Among these factors, copper is known to be the strongest enhancer of shikonin production. Although shikonin biosynthesis has been studied for over 40 years, the steps of naphthalene ring formation are still unknown, as is the reason for the effect of copper. In this study, we explored candidate genes associated with shikonin production using a PCR-select subtraction experiment. Polyphenol oxidase (PPO), a dicopper-dependent oxidoreductase, was highlighted because it showed synchronous expression with shikonin production. Transcriptome analysis of hairy roots and cultured cells of this plant revealed that, of the five PPO genes expressed in L. erythrorhizon , only PPO1 showed a strong correlation with shikonin production. Next, we generated genome-edited hairy roots of LePPO1 using CRISPR/Cas9-mediated mutagenesis to analyze its impact on shikonin derivative and other specialized metabolite production. The results showed that shikonin content was markedly reduced in all LePPO1 -ge lines. Interestingly, the content of deoxyshikonofuran, a hydroquinone derivative and shunt product that branches after GHQ-3′′-OH in the shikonin biosynthetic pathway, remained unaffected in the LePPO1 -ge lines. These findings suggest that LePPO1 participates in naphthalene ring formation and explain why a copper ion is crucial for shikonin biosynthesis.

ABSTRACT Down Syndrome (DS) is the most abundant genetic form of mental retardation. It is caused by the triplication of partial or complete human chromosome 21 (HSA21). The molecular mechanisms causing it are not fully understood. Previous studies identified “Down syndrome Critical Region” (DSCR) genes that are essential or sufficient for the development of DS. However, these studies are largely inconclusive, due, in part, to the reliance on a small number of epidemiological cases. Amyloid precursor protein ( APP ) resides on HSA21 and is triplicated in DS. APP plays a role in developmental and post-natal neurogenesis, but is not thought to be part of the DSCR. The role of APP overdose in cortical malformation and cognitive impairments in DS is unknown. Mutations in APP cause familial Alzheimer’s disease (FAD). However, whether APP overdose is sufficient for the development of Alzheimer’s disease (AD) in DS is not fully understood. Here, we addressed the role of APP overdose in neuronal development and AD pathology. Using CRISPR/Cas9 gene editing, we eliminated one copy of APP from Down Syndrome-derived induced iPSCs DS APP(+/+/-) and examined the effect on neurogenesis, AD-related pathology and the expression levels of genes on HSA21 that are implicated in DS, neurodegeneration and inflammation.

Gene knock-in therapy has the potential to cure inherited liver diseases but is limited by low efficiency and delivery complexity. Here, we developed a single adeno-associated virus (AAV) vector system comprising a compact CRISPR effector, enAsCas12f, a guide RNA, and a donor template to enable therapeutic genome editing via non-homologous end joining (NHEJ). We targeted the system to the murine Alb locus and applied it to mouse models of hemophilia B, protein C (PC) deficiency, and ornithine transcarbamylase (OTC) deficiency. NHEJ-mediated knock-in showed higher efficiency than homology-directed repair, with successful therapeutic gene insertion in both neonatal and adult mice. The strategy restored plasma factor IX activity in hemophilia B ( F9 −/− ) mice, prolonged survival of PC-deficient ( Proc −/− ) mice, and prevented hyperammonemia and weight loss in OTC-deficient ( Otc spf-ash ) mice upon high protein challenge. Importantly, gene integration was restricted to the liver, with no evidence of germline transmission. This compact, all-in-one AAV knock-in platform simplifies vector production, enables efficient delivery, and achieves reliable transgene expression in vivo . Our findings highlight the potential of liver-targeted knock-in genome editing as a transplant-independent treatment for neonatal-onset metabolic diseases, offering a clinically feasible path towards curative gene therapies for a wide range of monogenic liver disorders.

Summary Background 4-1BB (CD137), a member of the TNF receptor superfamily, is a critical co-stimulatory receptor for CD8⁺ T cell activation and regulatory T cell (Treg) expansion. While its ligand 4-1BBL is typically expressed by professional antigen-presenting cells, several carcinomas also express 4-1BBL, though its function in the tumor microenvironment remains poorly defined. Methods We analyzed 4-1BBL expression across human tumors and found papillary renal cell carcinoma (pRCC) to exhibit the highest levels. Using The Cancer Genome Atlas, we found high 4-1BBL expression correlated with poor overall survival in pRCC. To study its role in vivo, we established an orthotopic humanized mouse model of pRCC by grafting ACHN cells into the renal capsule of mice reconstituted with human CD34⁺ hematopoietic stem cells. We then performed CRISPR-mediated deletion of 4-1BBL in tumor cells, followed by flow cytometry and single-cell RNA sequencing of tumor-infiltrating immune cells. Results Loss of tumor-derived 4-1BBL resulted in accelerated tumor growth and decreased immune cell clustering. In the absence of 4-1BBL, CD8⁺ T cells displayed elevated expression of PD-1, TIM-3, LAG-3, granzyme B, perforin, and NKG7, indicating a cytotoxic yet exhausted phenotype. Treg were only modestly impacted. Tumor-infiltrating CD8⁺ T cells expressed high levels of 4-1BBL and showed transcriptional signatures of altered AP-1 factors and enhanced PI3K pathway signaling. Conclusions Our findings uncover a previously unrecognized role for tumor- and T cell–derived 4-1BBL in sustaining cytotoxic CD8⁺ T cell functionality and limiting their exhaustion. This reveals a potential immune-regulatory axis that could be exploited for therapeutic modulation in renal cell carcinoma.

Johanson-Blizzard Syndrome (JBS) is an autosomal recessive spectrum disorder associated with the UBR-1 ubiquitin ligase that features developmental delay including motor abnormalities. Here, we demonstrate that C. elegans UBR-1 regulates high-intensity locomotor behavior and developmental viability via both ubiquitin ligase and scaffolding mechanisms. Super-resolution imaging with CRISPR-engineered UBR-1 and genetic results demonstrated that UBR-1 is expressed and functions in the nervous system including in pre-motor interneurons. To decipher mechanisms of UBR-1 function, we deployed CRISPR-based proteomics using C. elegans which identified a cadre of glutamate metabolic enzymes physically associated with UBR-1 including GLN-3, GOT-2.2, GFAT-1 and GDH-1. Similar to UBR-1, all four glutamate enzymes are genetically linked to human developmental and neurological deficits. Proteomics, multi-gene interaction studies, and pharmacological findings indicated that UBR-1, GLN-3 and GOT-2.2 form a signaling axis that regulates glutamate homeostasis. Developmentally, UBR-1 is expressed in embryos and functions with GLN-3 to regulate viability. Overall, our results suggest UBR-1 is an enzyme hub in a GOT-2.2/UBR-1/GLN-3 axis that maintains glutamate homeostasis required for efficient locomotion and organismal viability. Given the prominent role of glutamate within and outside the nervous system, the UBR-1 glutamate homeostatic network we have identified could contribute to JBS etiology.

Toxoplasma and other Apicomplexan parasites, switch between different developmental stages to persist in and transmit between hosts. Toxoplasma can alternate between systemic tachyzoites and encysted bradyzoite forms found in the CNS and muscle tissues. How parasites sense these tissue types and trigger differentiation remains largely unknown. We show that Toxoplasma differentiation is induced under glucose-limiting conditions and using a CRISPR screen identify parasite genes required for growth under these conditions. From ∼25 identified genes important for differentiation we show that lactate and glutamine metabolism is linked to differentiation and demonstrate the importance of an E3 ubiquitin ligase complex, orthologous to glucose induced degradation deficient (GID) complex in yeast and CTLH complex in humans. We show that TgGID likely regulates translational repression of a key transcription factor required for differentiation, BFD1, through its 3’ utr. Overall, this work provides important new insight into how these divergent parasites sense different host cell niches and trigger stage conversion through a ubiquitination-dependent program.

Abstract It was recently shown that inhibition of polo-like kinase 4 (PLK4) induces TP53 -dependent synthetic lethality in cancers with chromosome 17q-encoded TRIM37 copy number gain due to cooperative regulation of centriole duplication and mitotic spindle nucleation. We show here that chromosome 17q/TRIM37 gain is a pathognomonic feature of high-risk neuroblastoma and renders patient-derived cell lines hypersensitive to the novel PLK4 inhibitor RP-1664. We demonstrate that centriole amplification at low doses of RP-1664 contributes to this sensitivity in a TRIM37 - and TP53 -independent fashion. CRISPR screens and live cell imaging reveal that upon centriole amplification, neuroblastoma cells succumb to multipolar mitoses due to an inability to cluster or inactivate supernumerary centrosomes. RP-1664 showed robust anti-tumor activity in 14/15 neuroblastoma xenograft models and significantly extended survival in a transgenic murine neuroblastoma model. These data support biomarker-directed clinical development of PLK4 inhibitors for high-risk neuroblastoma and other cancers with somatically acquired TRIM37 overexpression.

Abstract Despite initial responses, most patients with metastatic lung cancer—including those with EGFR mutations—ultimately develop resistance to targeted therapies. To systematically uncover mechanisms underlying this resistance, genome-wide CRISPR knockout and activation screens were conducted in EGFR-mutant lung cancer cell lines treated with EGFR inhibitors such as osimertinib and gefitinib. These screens highlighted a recurrent involvement of genes associated with the Hippo signaling pathway. Notably, a subset of tumor cells, termed 'persister' cells, survive initial osimertinib exposure by engaging non-genetic, transcriptional adaptation mechanisms that promote drug tolerance. Our studies, integrating both genetic and pharmacological approaches, identified Hippo pathway activation as a key driver of this drug-tolerant state. Importantly, co-inhibition of EGFR and the Hippo signaling axis led to a pronounced reduction in cell viability in both established cell lines and patient-derived organoids. These findings propose that dual targeting of EGFR and Hippo signaling may offer a promising therapeutic approach to overcome resistance in EGFR-mutant lung cancer.

Objectives: To explore the molecular chemistry and structural biology of bacteriophages as precision-guided therapeutic agents. This review reframes phages as programmable nanomachines governed by defined chemical interactions, focusing on their relevance to antimicrobial resistance, targeted lysis, and synthetic modification. Materials and Methods: A systematic review was conducted using recent peer-reviewed literature from PubMed, Scopus, and Google Scholar, covering bacteriophage structural biology, enzymatic lysis mechanisms, chemical modifications, genomic annotation, and bioengineering techniques. Studies were selected based on relevance to molecular chemistry, nanotechnology, and therapeutic development. Results: Bacteriophages demonstrate ligand-specific host recognition, capsid-mediated genome packaging, and enzymatic lysis using holins and endolysins. Advances in PEGylation, surface conjugation, and CRISPR engineering have expanded their therapeutic potential. Genomic tools now enable personalized phage matching, while hybrid phage-nanoparticle systems enhance targeting and delivery. Conclusions: Phages can be rationally designed as chemically programmable antimicrobials. Their structure–function relationship, enzymatic precision, and genomic adaptability position them as promising agents in the fight against multidrug-resistant pathogens. Integration of chemistry, bioinformatics, and synthetic biology enables development of next-generation phage therapeutics.