Bulk lipid transport between organelles has been proposed to involve the partnership between bridge lipid transport proteins and membrane-embedded lipid scramblases. However, for almost all BLTPs, such physical association has not been fully described, and, in most cases, the identity of the scramblases is unknown. Here, we identify TMEM170 family proteins as endoplasmic reticulum lipid scramblases that physically interact with BLTP1/Csf1 proteins. This finding opens new avenues to understand the complex mechanism involved in lipid transport at membrane contact sites.

Immune checkpoint inhibitor (ICB) therapy for many cancers remains limited in patients’ overall response rate. Discovery and development of more effective combinatorial approaches is urgent. Here, through CRISPR/Cas9 genetic screens, we identify DOT1L as a versatile epigenetic factor that functions to suppress tumor-intrinsic immunity through a dual mechanism. Depletion of DOT1L induces the expression of transposable elements and subsequent type I interferon (IFN) response, and meanwhile lowers ZEB1 levels to further unleash the expression of immune-related genes. In turn, we demonstrate that DOT1L loss or treatment with the clinical stage inhibitor EPZ-5676 sensitizes tumors to ICB with increased immune infiltration in mice. More importantly, EPZ-5676 treatment alone is sufficient to enhance antitumor immunity in humanized mice. TCGA data analysis reveals an inverse correlation between DOT1L expression and IFN signatures across multiple cancer types. These findings provide a rationale for targeting DOT1L to improve tumor immunogenicity and overcome immunotherapy resistance. One Sentence Summary CRISPR genetic screens identified DOT1L as a potential suppressor of tumor intrinsic immunogenicity

Objective The accumulation of DNA damage and mutations is a key contributor to aging. Recent studies have shown that disrupting the Beclin 1-BCL2 autophagy regulatory complex through gene editing can extend lifespan in mice. The precise application of gene editing technologies offers a promising strategy for aging. This study conducted a bibliometric analysis to map the knowledge landscape of gene editing and aging. Methods We retrieved publications related to genome editing and aging from the Web of Science Core Collection, covering the period from 2015 to 2024. The data were analyzed using VOSviewer and R package Bibliometrix. These tools enabled us to identify the most productive researchers, journals, institutions, countries and visualized current trends, emerging research hotspots. Results A total of 982 publications on genome editing and aging were identified. The United States (n=285) and China (n=214) form a dual-core structure leading global output. Harvard University (n=116) emerged as the most prolific institution. Scientific Reports was the top-publishing journal, with 23 articles in 2024. ZHANG Y (n=12, citations=102, H-index=6) was identified as the most productive author. KIM E’s 2017 publication in Nature Communications (TC=494, TC/year=54.9, NTC=9.33) has had a significant and ongoing impact. The analysis indicates that future directions will include CRISPR optimization and AI-assisted genomic analysis. Conclusion This study presents the first comprehensive bibliometric analysis and visualization of the knowledge structure in gene editing and aging research up to 2024. It offers researchers a detailed overview of current developments, trends, and emerging frontiers in this rapidly evolving domain.

Neurons contribute to the complex interplay of signals that mediate heart development and homeostasis. Although a limited set of studies suggest that neuronal peptides impact vertebrate heart growth, the specific contributions of these peptides to cardiomyocyte progenitor differentiation or proliferation have not been elucidated. Here we show that the neuropeptide tachykinin along with canonical Wnt signaling regulate cardiomyocyte progenitor proliferation in the chordate model Ciona robusta . In C. robusta, the heart continues to grow throughout adulthood and classic histological studies indicate that a line of undifferentiated cells may serve as a reserve progenitor lineage. We found that this line of cardiomyocyte progenitors consists of distinct distal and midline populations. Distal progenitors divide asymmetrically to produce distal and midline daughters. Midline progenitors divide asymmetrically to produce myocardial precursors. Through single cell RNA sequencing (scRNA-seq) of adult C. robusta hearts, we delineated the cardiomyocyte progenitor expression profile. Based on this data we investigated the role of Wnt signaling in cardiomyocyte progenitor proliferation and found that canonical Wnt signaling is required to suppress excessive progenitor proliferation. The scRNA-seq data also identified a number of presumptive cardiac neural-like cells. Strikingly, we found that a subset of these neuronal cells appears to innervate the distal cardiomyocyte progenitors. Based on the expression of the tachykinin receptor in these neuronal cells, we blocked tachykinin signaling using pharmacological inhibitors and found that this drove reduced proliferation in the distal progenitor pool. Through targeted CRISPR-Cas9 knockdown we then demonstrated that both extrinsic tachykinin and intrinsic, cardiac tachykinin receptors are required for formation of the myocardial heart tube. This work provides valuable insights into how organisms may deploy neural signals to regulate organ growth in response to environmental or homeostatic inputs.

ABSTRACT Optogenetic tools – whose engineering requires a structural understanding of the target proteins – are attractive approaches for achieving endogenous gene regulation under minimally invasive conditions. To build an optogenetic system for controlling endogenous gene expression, we first identified Anti-CRISPR (Acr) proteins that can inhibit CRISPRa-mediated transcriptional activation in Drosophila . Next, we inserted optogenetic protein LOV2 in these Acrs, tested for their ability to optogenetically modulate endogenous gene upregulation (EnGup) through the CRISPRa-based flySAM system in Drosophila , and found that the photoswitchability of these prototypes was weak. Hence, we engineered an Acr-LOV2 fusion module with refined length of intrinsically disordered and ordered regions (IDR and IOR) and optimized LOV2. This optimized variant, whose application yielded new findings in vivo , was significantly more sensitive for EnGup under blue light than the prototypes. In short, not only does this work introduce the application of Acr proteins in an animal model, but it also provides insights for in vivo characterization of the IDR and the IOR of these small-sized proteins. Together, these findings establish a robust optogenetic toolbox for precise, light-sensitive endogenous gene regulation in Drosophila . Graphical abstract

The human skin microbiome is shaped by a complex interplay of host physiology, environmental exposure, and microbial interactions across domains of life. However, the relative contributions of host genetics and geography remain unresolved. Here, we present the first application of ultra-low coverage human genome imputation from skin metagenomic data, analysing 1,756 samples from multiple skin types and timepoints, with matched genotypes from 327 individuals across five countries. We also generate expanded Skin Microbial Genome Collection (eSMGC), comprising 675 prokaryotic, 12 fungal, 2,344 viral, and 4,935 plasmid genomes, correcting extensive false positives in existing references. Intercountry comparisons reveal that geography explains more microbiome variation than skin type, and that host genetics contributes previously uncharacterized structure—exemplified by distinct profiles in Chinese individuals. Genome-wide association analysis identifies 107 SNPs linked to 22 microbial taxa, including phages and plasmids, implicating host genes in skin structure, immunity, and lipid metabolism. Cutibacterium acnes and its phages exhibit geographic divergence and phage–host co-adaptation. Finally, host-infecting viruses, particularly papillomaviruses, are associated with elevated microbial diversity and immune-modulatory functions. These findings establish host genetics as a determinant of skin microbiome ecology and highlight the value of multi-domain, geographically diverse analyses.

Synthetic lethality describes a genetic relationship where the loss of two genes results in cell death, but the loss of one of those genes does not. Drugs used for precision oncology can exploit synthetic lethal relationships; the best described are PARP inhibitors which preferentially kill BRCA1 -deficient tumours preferentially over BRCA1 -proficient cells. New synthetic lethal targets are often discovered using genetic screens, such as CRISPR knockout screens. Here, we present a competitive co-culture assay that can be used to analyse drugs or gene knockouts with synthetic lethal effects. We generated new BRCA1 isogenic cell line pairs from both a triple-negative breast cancer cell line (SUM149) and adapted pre-existing non-cancerous BRCA1 isogenic pair (RPE). Each cell line of the isogenic pair was transformed with its own fluorescent reporter. The two-coloured cell lines of the isogenic pair were then grown together in the same vessel to create a more competitive environment compared to when grown separately. We used four PARP inhibitors to validate the ability to detect synthetic lethality in BRCA1 -deficient cancer cells. The readout of the assay was performed by counting the fluorescently coloured cells after drug treatment using flow cytometry. We observed preferential targeting of BRCA1- deficient cells, by PARPi, at relative concentrations that broadly reflect clinical dosing. Further we reveal subtle differences between PARPi resistant lines compared to BRCA1- proficient cells. Here, we demonstrate the validation and potential use of the competitive assay, which could be extended to validating novel genetic relationships and adapted for live cell imaging.

Thermogenetics enables non-invasive spatiotemporal control over protein activity in living cells and tissues, yet its applications have largely been restricted to transcriptional regulation and membrane recruitment. Here, we present a generalizable strategy for engineering thermosensitive allosteric proteins through the insertion of optimized Avena sativa LOV2 domain variants. Applying this approach to a diverse set of structurally and functionally unrelated proteins in Escherichia coli , we generated potent, thermo-switchable chimeric variants that can be tightly controlled within narrow temperature ranges (37-41°C). Extending this strategy to mammalian systems, we engineered the first CRISPR-Cas genome editors directly modulated by subtle temperature changes within the physiological range. Finally, we showcase the incorporation of a chemoreceptor domain as an alternative thermosensing module, suggesting thermo-sensitivity to be a widespread feature in receptor domains. This work expands the toolkit of thermogenetics, providing a blueprint for temperature-dependent control of virtually any protein of interest.

ABSTRACT Lentiviral vectors are a cornerstone delivery modality of biomedical research, renowned for their ability to stably integrate genetic material into the host genome, enabling sustained transgene expression and long-term genetic manipulation. These properties make them indispensable tools in functional genomics and genome engineering, particularly for delivering molecular components in high-throughput CRISPR screening, a powerful approach for uncovering the genetic basis of complex cellular mechanisms and phenotypes. However, challenges such as lentiviral-induced recombination, unpredictable integration profiles, and variable susceptibility of target cells to transduction can introduce noise and compromise experimental outcomes. In this study, we selected two suspension-adapted mammalian cell lines, Chinese Hamster Ovary cells CHO-K1 and Human Embryonic Kidney cells HEK293-6E, due to their widespread use in recombinant protein production. Recognizing the influence of intrinsic cell line properties and transduction methodology, we compared two distinct procedures: spinoculation and static transduction. By implementing a two-step static transduction protocol, we achieved significantly higher transduction efficiencies while minimizing cellular stress, streamlining workflows, and eliminating scalability limitations inherent to large-scale lentiviral applications like genome-wide CRISPR screens. To further characterize the variation in lentiviral integration, we used droplet digital PCR (ddPCR) to quantify copy number variation (CNV) both at the pooled population level and within individual clonal isolates. This comprehensive analysis underscores the robustness of our optimized protocol in enhancing transduction efficiency in difficult-to-transduce suspension cell lines. It further emphasizes the importance of carefully modulating infection rates to limit multiple integrations, ensuring the accuracy and consistency required for large-scale functional genomics applications.

ABSTRACT Ethanol is a fermentation product widely used as a fuel and chemical precursor in various applications. However, its accumulation imposes severe stress on the microbial producer, leading to significant production losses. To address this, improving a strain’s ethanol tolerance is considered an effective strategy to enhance production. In our previous research, we conducted an adaptive evolution experiment with Escherichia coli growing under gradually increasing concentrations of ethanol, which gave rise to multiple hypertolerant populations. Based on the genomic mutational data, we demonstrated in this work that adaptive alleles in the EnvZ-OmpR two-component system drive the development of ethanol tolerance in E. coli . Specifically, when a single leucine was substituted for a proline residue within the periplasmic domain using CRISPR, the mutated EnvZ osmosensor caused a significant increase in ethanol tolerance. Through promoter fusion assays, we showed that this particular mutation stabilizes EnvZ in a kinase-dominating state, which reprograms signal transduction involving its cognate OmpR response regulator. Whole-genome proteomics analysis revealed that this altered signaling pathway predominantly maintains outer membrane stability by upregulating global porin levels and attenuating iron metabolism in the tolerant envZ* L116P mutant. Moreover, we demonstrated that the hypertolerant envZ* L116P allele also promotes ethanol productivity in fermentation, providing valuable insights for enhancing industrial ethanol production. AUTHOR SUMMARY Ethanol is a versatile chemical with many applications, but producing it in high quantities remains a challenge. This is because Escherichia coli , a candidate ethanol production strain, is naturally sensitive to this short-chain alcohol, especially when levels are gradually accumulating during fermentation. To resolve this bottleneck, we have investigated how E. coli can acquire tolerance to its own toxic fermentation product. Our research indicated that a single amino acid substitution in EnvZ-a key sensor protein that normally protects E. coli against extreme osmotic stress-is sufficient to confer ethanol tolerance. Further analysis revealed that the mutation perturbs the EnvZ-mediated signaling cascade, which, in turn, changes the transporter composition in the outer membrane and attenuates the cell’s iron metabolism. These adaptations enable E. coli to survive under high-ethanol conditions, thereby promoting its ethanol production efficiency. This discovery provides a suitable strategy to increase ethanol titers in industrial settings using fermentation.

Background Metastatic colorectal cancer (mCRC) is associated with high recurrence rates and resistance to conventional treatments, largely driven by cancer stem cells (CSCs) that contribute to tumor progression and therapeutic evasion. This study aims to investigate the role of netrin-1 and its dependence receptor UNC5B in regulating CSC self-renewal in mCRC and explore their potential as therapeutic targets. Methods We used patient-derived liver metastasis organoids (PDOs) to examine the effects of netrin-1 on CSC self-renewal. The role of UNC5B was evaluated by silencing its expression using CRISPR and assessing the impact on CSC apoptosis in response to an anti-netrin-1 blocking antibody (NP137) using extreme limiting dilution assays (ELDAs). Single-cell RNA sequencing was employed to explore the molecular mechanisms behind netrin-1/UNC5B regulation of CSC fate. Clinical data from a patient with mCRC were used to validate the findings. Results Netrin-1 promoted CSC self-renewal by inhibiting apoptosis, a process reversed by NP137. UNC5B was identified as the primary receptor mediating this effect, as its silencing eliminated Netrin-1-induced self-renewal. Trefoil Factor 3 (TFF3), secreted by UNC5B-expressing cells, plays a key role in netrin-1-induced CSC self-renewal. Clinical trial data from a patient with mCRC showed a reduction in TFF3 and stemness genes expression after treatment with NP137. Furthermore, combining NP137 with FOLFOX chemotherapy enhanced cell death and inhibited tumor growth in PDO xenograft models. Conclusion This study identifies the netrin-1/UNC5B/TFF3 axis as a critical regulator of CSC self-renewal in mCRC and suggests that targeting this pathway with NP137, in combination with chemotherapy, could provide a promising therapeutic approach for mCRC patients.

Despite major advances in genetic screening technology, a formal approach for quantifying gene function remains underdeveloped, thereby limiting the utility of these techniques in deciphering the complex behavior of human cells. In this study, we leverage information theory with a perturbational analysis of replicator dynamics to characterize functional drivers of selection in pooled CRISPR screens. Our approach challenges established methods for CRISPR screen analysis, while offering additional insight into selection dynamics through the Kullback-Leibler divergence ( D KL ) and cumulants of the fitness distribution. By modeling fluctuations in gene-fitness effects as a linear response to environmental perturbations, we derive a geometric measure for genomic information content based on a second-order approximation of the D KL . Our analysis reveals that functional information—encoded (or shared) between genes—can be quantified by analyzing the directions corresponding to maximal conditional selection within the space of decomposed gene-environment interactions. This geometric representation offers several advantages for the functional analysis of the human genome and its network architecture. Moreover, by constraining the space to cell-type-specific fluctuations, we uncover developmental and tissue-specific functional signatures. These findings represent significant progress in the dynamic analysis of gene function and in the functional wiring of the human genome.

Human brain development is highly regulated by several spatiotemporal processes, which disruption can result in severe neurological disorders. Emerging evidence highlights the pivotal role of mitochondrial function as one of these fundamental pathways involved in neurodevelopment. Our study investigates the role of 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) protein in cortical neurogenesis and mitochondrial activity, since mutations in the HPDL gene are associated with SPG83, a childhood-onset form of hereditary spastic paraplegia characterized by corticospinal tract degeneration and cortical abnormalities. Starting from mutant neuroblastoma cells, we demonstrated that HPDL is essential to mitochondrial respiratory chain supercomplex assembly and cellular redox balance. Moreover, transcriptomic analyses revealed dysregulated pathways related to neurogenesis, implicating HPDL role in early cortical development. To further elucidate the role of HPDL, we generated cortical neurons and organoids from SPG83 patient-derived induced pluripotent stem cells. Mutant cells exhibited premature neurogenesis at early differentiation stages, likely leading to depletion of cortical progenitors, as evidenced by decreased proliferation, slight increase of apoptosis, and unbalanced cortical type composition at later stages. Furthermore, cortical organoids derived from SPG83 patients showed impaired growth, reminding microcephaly observed in severe cases. In addition, mitochondrial morpho-functional characterization in mutant neurons confirmed disruption of OxPhos chain functionality and increased ROS generation rate. Treatment of cortical cells with two antioxidant compounds, could partially revert premature neurogenesis. In conclusion, our findings reveal a critical role for HPDL in coordinating cortical progenitor proliferation, neurogenesis, and mitochondrial function. These insights shed light on a mechanistical understanding of SPG83 pathology and underscore the therapeutic potential of targeting oxidative stress in this and related neurological disorders.

ABSTRACT Zinc-finger Antiviral Protein (ZAP)-mediated RNA decay (ZMD) restricts replication of viruses containing CpG dinucleotide clusters. However, why ZAP isoforms differ in antiviral activity and how they recruit cofactors to mediate RNA decay is unclear. Therefore, we determined the ordered events of the ZMD pathway. The long ZAP isoform preferentially binds viral RNA, which is promoted by TRIM25. The endoribonuclease KHNYN then cleaves viral RNA at positions of ZAP binding. The 5’ cleavage fragment undergoes TUT4/TUT7-mediated 3’ uridylation and degradation by DIS3L2. The 3’ cleavage fragment is degraded by XRN1. ZAP and TRIM25 interact with KHNYN, TUT7, DIS3L2 and XRN1 in a RNase-resistant manner. Viral infection promotes the interaction between ZAP and TRIM25 with these enzymes, leading to viral RNA degradation while also decreasing the abundance of many cellular transcripts. Overall, the long isoform of ZAP recruits key enzymes to assemble an RNA decay complex on viral RNA.

Candida parapsilosis is an opportunistic yeast pathogen that can cause life-threatening infections in immunocompromised humans. Whole genome sequencing (WGS) studies of the species have demonstrated remarkably low diversity, with strains typically differing by about 1.5 single nucleotide polymorphisms (SNPs) per 10 kb. However, SNP calling alone does not capture the full extent of genetic variation. Here, we define the pangenome of 372 C. parapsilosis isolates to determine variation in gene content. The pangenome consists of 5,859 genes, of which 48 are not found in the genome of the reference strain. This includes 5,791 core genes (present in ≥ 99.5% of isolates). Four genes, including the allantoin permease gene DAL4 , were present in all isolates but were truncated in some strains. The truncated DAL4 was classified as a pseudogene in the reference strain CDC317. CRISPR-Cas9 gene editing showed that removing the early stop codon (producing the full-length Dal4 protein) is associated with improved use of allantoin as a sole nitrogen source. We find that the accessory genome of C. parapsilosis consists of 68 homologous clusters. This includes 38 previously annotated genes, 27 novel paralogs of previously annotated genes and 3 uncharacterised ORFs. Approximately one-third of the accessory genome (24/68 genes) is associated with gene fusions between tandem genes in the major facilitator superfamily (MFS). Additionally, we identified two highly divergent C. parapsilosis strains and find that, despite their increased phylogenetic distance (∼30 SNPs per 10 kb), both strains have similar gene content to the other 372. Importance Candida parapsilosis is a human fungal pathogen, listed in the high priority group by the World Health Organisation. It is an increasing cause of hospital-acquired and drug-resistant infection. Here, we studied the genetic diversity of 372 C. parapsilosis isolates, the largest genomic surveillance of this species to date. We show that there is relatively little genetic variation. However, we identified two more distantly-related isolates from Germany, suggesting that even more sampling may yield more diversity. We find that the pangenome (the cumulative gene content of all isolates) is surprisingly small, compared to other fungal species. Many of the non-core genes are involved in transport. We also find that variations in gene content are associated with nitrogen metabolism, which may contribute to the virulence characteristics of this species.

Genetic functional screening technologies which identify causative genes are essential for advancing life sciences and improving drug discovery outcomes. Traditional array-based screening methods, which require significant cell numbers, face limitations when working with samples that have low proliferation capacity. While pooled library methods such as CRISPR screens can be solutions to these experimental efficiency challenges, there is still room for improvement in terms of cost and convenience. In response to these challenges, we developed PiER (Perturbation-induced intracellular events recorder) technology. PiER facilitates gene perturbation and intracellular signal detection through a novel system that integrates three DNA domains. The Perturbation domain induces gene-specific disturbances, the Response domain expresses an enzyme upon desired cellular signals, and the Memory domain records perturbation history by altering its DNA sequence via the expressed enzyme. To demonstrate PiER’s potential, we designed a vector which has a Response domain that detects WNT pathway activation. Transfecting HEK293 cells, we observed dose-dependent responses to WNT pathway activation using fluorescence microscopy and quantitative Polymerase Chain Reaction (qPCR), which confirmed successful intracellular event recording in the Memory domain. Further experiments with lentiviral PiER vectors containing a pooled shRNA library revealed the system’s capability to conduct high-throughput screening by analyzing perturbations and their effects within individual cells. PiER technology significantly enhances screening capabilities by offering a versatile and scalable approach that can be deployed without prior cell modification and single-cell isolation. Its high throughput, combined withrequiring minimal effort, presents a significant advancement for genomic research and drug target discovery.

The 7SK snRNP is a ribonucleoprotein complex comprising the non-coding RNA 7SK and the associated proteins MePCE, LARP7, and HEXIM. It regulates transcription in higher eukaryotes by sequestering the positive transcription elongation factor (P-TEFb), preventing premature entry of RNA Polymerase II in elongation. Loss of LARP7 in humans causes the Alazami syndrome, marked by restricted growth, impaired movement, and intellectual disability, though the underlying mechanisms remain unclear. In this study, we show that loss of Larp7 or 7SK RNA in Drosophila is viable but impairs locomotion and reduces axonal growth at neuromuscular junctions. Larp7 is enriched in specific motoneurons, where it functions autonomously to promote axogenesis. Reducing P-TEFb abundance partially rescues the locomotion and axonal growth defects, indicating that the 7SK complex mediates this function via transcriptional regulation. Transcriptomic analysis of mutant motoneurons revealed that the 7SK complex primarily regulates long genes with high GC content at their promoters. These findings provide new insights into the tissue-specific roles of the 7SK snRNP in transcription and organismal function.

Comparative genomic studies between contemporary and extinct hominins revealed key evolutionary modifications, but their number has hampered a system level investigation of their combined roles in scaffolding modern traits. Through multi-layered integration we selected 15 genes carrying nearly fixed sapiens -specific protein-coding mutations and developed a scalable design of combinatorial CRISPR-Cas9 bidirectional perturbations to uncover their regulatory hierarchy in cortical brain organoids. Interrogating the effects of overexpression and downregulation for all gene pairs in all possible combinations, we defined their impact on transcription and differentiation and reconstructed their regulatory architecture. We uncovered marked cell type-specific effects, including the promotion of alternative fates and the emergence of interneuron populations, alongside a core subnetwork comprising KIF15 , NOVA1 , RB1CC1 and SPAG5 acting as central regulator across cortical cell types.

The ubiquitin-proteasome system (UPS) is a fundamental regulatory mechanism maintaining cellular proteostasis through the targeted degradation of proteins. Beyond its canonical role in protein turnover, the UPS governs diverse biological processes, including cell cycle control, DNA repair, immune responses, and stress adaptation. Dysregulation of UPS components is increasingly recognized as a driving force in the pathogenesis of numerous diseases, such as cancer, neurodegenerative disorders, metabolic dysfunctions, and infections. In plants, the UPS also plays a pivotal role in environmental stress responses and hormone signaling, offering promising avenues for crop improvement. This review presents a comprehensive overview of the molecular architecture and functions of the UPS, explores its role in maintaining cellular and systemic homeostasis, and critically examines the consequences of UPS dysfunction across various disease contexts. We further highlight emerging technologies, including ubiquitinomics, CRISPR-based screens, and targeted protein degradation platforms, that are accelerating UPS research. Finally, we discuss current challenges and future opportunities for translating UPS insights into therapeutic and biotechnological innovations. A deeper understanding of the UPS across biological systems is essential for developing next-generation strategies to combat human diseases and enhance agricultural resilience.

ABSTRACT Over the past decade, Immuno-Oncology has largely focused on blocking inhibitory surface receptors like PD-1 to enhance T cell anti-tumor activity. However, intracellular immune checkpoints such as CISH, which function independently of tumor-expressed ligands, offer powerful and previously untapped therapeutic potential. As a downstream regulator of TCR signaling, CISH controls T cell activation, expansion, and neoantigen reactivity. Though historically considered undruggable, recent advances in CRISPR engineering have enabled functional interrogation of these targets. We demonstrate that CISH deletion enhances T cell activation and anti-cancer functions more effectively than other emerging intracellular checkpoints. In CAR-T cells, CISH inactivation significantly increased sensitivity to tumor antigen, enabling robust recognition and killing even at low antigen levels, conditions that often lead to treatment failure with conventional T cell therapies, mirroring antigen escape scenarios seen in solid tumors. Our findings further validate CISH as a potent and druggable intracellular checkpoint capable of boosting anti-tumor T cell responses across diverse cancer types, independent of PD-L1 status. The underlying mechanisms of CISH inhibition may help explain the positive outcomes reported in recent clinical studies of this approach in solid tumor immunotherapy.

Glandular trichomes are specialized epidermal structures that play an essential role in plant defense by synthesizing, storing, and secreting specialized metabolites. This study investigates the function of NtAGL66 , an AGAMOUS-like gene in Nicotiana tabacum , uncovering its role in the development of secretory heads in long glandular trichomes. Expression profiling reveals that NtAGL66 is specifically expressed in the developing secretory glands. Functional analyses show that NtAGL66 overexpression promotes the differentiation of the secretory structure, while CRISPR-Cas9-mediated knockout significantly reduces the capacity of trichomes to form functional secretory glands, highlighting its essential role in trichome specialization. Transcriptomic (RNA-seq) and functional genomic (DAP-seq) analyses indicate that NtAGL66 regulates also secondary metabolic pathways and is likely involved in broader transcriptional networks, including floral development. Notably, this includes genes such as NtTOE1, previously shown to control both floral organogenesis and glandular trichome formation in tomato. Moreover, NtAGL66 directly regulates the transcription factor NtGL2 through promoter binding. By identifying an AGAMOUS-like gene as a key regulator of secretory gland development, this study offers novel insights into the genetic mechanisms underlying glandular trichome differentiation and specialized metabolite biosynthesis in Solanaceae .

Summary CRISPR-Cas systems provide adaptive immunity against phage infection in prokaryotes using an RNA-guided complex that recognizes complementary foreign nucleic acids. Different types of CRISPR-Cas systems have been identified that differ in their mechanism of defense. Upon infection, Type III CRISPR-Cas systems employ the Cas10 complex to find phage transcripts and synthesize cyclic oligo-adenylate (cOA) messengers. These ligands bind and activate CARF immune effectors that cause cell toxicity to prevent the completion of the viral lytic cycle. Here we investigated two proteins containing an N-terminal haloacid dehalogenase (HAD) phosphatase domain followed by four predicted transmembrane helices and a C-terminal CARF domain, which we named Chp. We show that, in vivo, Chp localizes to the bacterial membrane and that its activation induces a growth arrest, leads to a depletion of ATP and IMP and prevents phage propagation during the type III CRISPR-Cas response. In vitro, the CARF domain of Chp binds cyclic tetra-adenylates and the HAD phosphatase domain dephosphorylates dATP, ATP and IMP. Our findings extend the range of molecular mechanisms employed by CARF effectors to defend prokaryotes against phage infection.

The natural context in which CRISPR-Cas systems are active in Enterobacteriaceae has remained enigmatic. Here, we find that the Citrobacter rodentium Type I-E CRISPR-Cas system is activated by the oxygen-responsive transcriptional regulator Fnr in the anoxic mouse intestine. Since Fnr-dependent regulation is predicted in ~41% of Enterobacteriaceae cas3 orthologs, we propose that anoxic regulation of CRISPR-Cas immunity is an adaptation that protects Enterobacteriaceae against threats arising from the intestinal microbiome.

Many taxa have independently evolved genetic sex determination where a single gene located on a sex chromosome controls gonadal differentiation. The gene anti-Mullerian hormone ( amh ) has convergently evolved as a sex determination gene in numerous vertebrate species, but how this gene has repeatedly evolved this novel function is not well understood. In the threespine stickleback ( Gasterosteus aculeatus ), amh was duplicated onto the Y chromosome ( amhy ) ∼22 million years ago. To determine whether amhy is the primary sex determination gene, we used CRISPR/Cas9 and transgenesis to show that amhy is necessary and sufficient for male sex determination, consistent with the function of a primary sex determination gene. Despite being indispensable for sex determination, we detected low levels of amhy expression throughout early development. This indicates the mechanism of sex determination is likely unrelated to overall dosage of amhy and its autosomal paralog, amha . Threespine stickleback have striking differences in behavior and morphology between sexes. The creation of sex reversed lines allow us to investigate the genetic basis of secondary sex characteristics. Here we show one of the classic traits important for reproductive success, male nuptial coloration, is controlled by both Y-linked genetic factors as well as hormonal factors independent of sex chromosome genotype. This research establishes stickleback as a model to investigate how amh regulates gonadal development and how this gene repeatedly evolves novel function in sex determination. Analogous to the four core genotypes model in house mice, sex-reversed threespine stickleback offer a new vertebrate model for investigating the separate contributions of gonadal sex and sex chromosomes to sexual dimorphism.

The typhoid toxin is a secreted virulence factor of typhoidal serovars of the bacterial pathogen Salmonella enterica implicated in typhoid fever and chronic infections. The toxin causes a DNA damage response in human cells, characterised by cell-cycle arrest and cellular distension, resulting in cellular senescence and increased bacterial burden. To better understand host responses to typhoid toxin, we performed a transcriptomic analysis of intoxicated host cells and found that the toxin induced expression of genes relating to the type-I interferon response, including the ubiquitin-like protein ISG15. ISG15 was upregulated in a STING-dependent manner, reduced bacterial burden, and was found to be critical to host cell survival in response to the typhoid toxin and purified interferon. This highlights ISG15 as an important component of the host cell defence to the typhoid toxin.