The levels of reactive oxygen species (ROS) and the extent of ensuing DNA damage significantly influence cancer initiation and progression. Of crucial importance, the aspartate protease DDI2 has been proposed to play a pivotal role in monitoring intracellular ROS levels (to trigger oxidative eustress or distress), as well as in the oxidative DNA damage repair, through redox homeostasis-determining factor Nrf1 (encoded by NFE2L1 ). However, the specific role of DDI2 in the multi-step process resulting in the development and progression of liver cancer remains elusive to date. In the present study, we employed the CRISPR/Cas9 gene editing system to create two nuanced lines of DDI2 knockout (i.e., DDI2 −/− and DDI2 insG/− ) from liver cancer cells. Subsequent experiments indicate that the knockout of DDI2 leads to increased ROS levels in hepatoma cells by downregulating two major antioxidant transcription factors Nrf1 and Nrf2 (encoded by NFE2L2 ), exacerbating endogenous DNA damages caused by ROS and not-yet-identified factors, thereby inhibiting cell proliferation and promoting apoptosis, and ultimately hindering in vivo malignant growth of xenograft tumor cells. Conversely, the restoration of DDI2 expression reverses the accumulation of ROS and associated DNA damage caused by DDI2 knockout, eliminating the subsequent inhibitory effects of DDI2 deficiency on both in vitro and in vivo growth of liver cancer cells. Collectively, these findings demonstrate that DDI2 deficiency impedes liver tumor growth by disrupting its survival environment, suggesting that DDI2 may serve as a novel therapeutic target for anti-cancer strategies aimed at modulating ROS or DNA damage processes.

Recent studies indicate that the development of drug resistance and increased invasiveness in melanoma is largely driven by transcriptional plasticity rather than canonical coding mutations. Understanding the mechanisms behind cell identity shifts in oncogenic transformation and cancer progression is crucial for advancing our understanding of melanoma and other aggressive cancers. While distinct melanoma phenotypic states have been well characterized, the processes and transcriptional controls that enable cells to shift between these states remain largely unknown. In this study, we initially leverage the well-established zebrafish melanoma model as a high-throughput system to dissect and analyze transcriptional control elements that are hijacked by melanoma. We identify key characteristics of these elements, making them translatable to human enhancer identification despite the lack of direct sequence conservation. Building on our identification of a zebrafish sox10 enhancer necessary for melanoma initiation, we extend these findings to human melanoma, identifying two human upstream enhancer elements that are critical for full SOX10 expression. Stable biallelic deletion of these enhancers using CRISPR-Cas9 induces a distinct phenotype shift across multiple human melanoma cell lines from a melanocytic phenotype towards an undifferentiated phenotype and is also characterized by an increase in drug resistance that mirrors clinical data including an upregulation of NTRK1, a tyrosine kinase, and potential therapeutic target. These results provide new insights into the transcriptional regulation of SOX10 in human melanoma and underscore the role of individual enhancer elements and potentially NTRK1 in driving melanoma phenotype plasticity and drug resistance. Our work lays the groundwork for future gene-based and combination kinase-inhibitor therapies targeting SOX10 regulation and NTRK1 as a potential avenue for enhancing the efficacy of current melanoma treatments.

Exposure to saturated fatty acids (SFAs), such as palmitic acid, can lead to cellular metabolic dysfunction known as lipotoxicity. Although canonical adaptive metabolic processes like lipid storage or desaturation are known cellular responses to saturated fat exposure, the link between SFA metabolism and organellar biology remains an area of active inquiry. We performed a genome-wide CRISPR knockout screen in human epithelial cells to identify modulators of SFA toxicity. The screen revealed peroxisomal proteins, especially those that impact ether lipid synthesis, as important regulators of lipotoxicity. We identified Fas-associated factor family member 2 (FAF2) as a critical bifunctional co-regulator of peroxisomal and fatty acid biology. We further uncovered a new biological function for the ubiquitin-regulatory X (UBX) and UAS thioredoxin-like domains of FAF2, demonstrating their requirement for peroxisomal protein abundance and SFA-induced cellular stress. Our work highlights the role of FAF2 in regulating peroxisomal abundance and function, and the peroxisome as a key organelle in the cellular response to SFAs.

WNK family kinases are regulated by osmotic stress and control ion homeostasis by activating SPAK and OXSR1 kinases. Using a proximity ligation approach, we found that osmotic stress promotes the association of WNK1 with the NRBP1 pseudokinase and TSC22D2/4 adaptor proteins, results that are confirmed by immunoprecipitation and mass spectrometry and immunoblotting studies. NRBP1 pseudokinase is closely related to WNK isoforms and contains a RΦ-motif binding conserved C-terminal (CCT) domain, similar to the CCT domains in WNKs, SPAK and OXSR1. Knockdown or knock-out of NRBP1 markedly inhibited sorbitol-induced activation of WNK1 and downstream components. We demonstrate recombinant NRBP1 can directly induce the activation of WNK4 in vitro . AlphaFold-3 modelling predicts that WNK1, SPAK, NRBP1, and TSC22D4 form a complex, in which two TSC22D4 RΦ-motifs interact with the CCTL1 domain of WNK1 and the CCT domain of NRBP1. Our data indicates NRBP1 functions as an upstream activator of the WNK pathway. Teaser NRBP1 functions as a scaffolding component regulating the assembly of a multi-subunit complex, required for the activation of the WNK Lysine Deficient Protein Kinase family in response to osmotic stress. Graphical Abstract

The increasing prevalence of multi-drug-resistant (MDR) bacterial pathogens presents a critical global health threat, highlighting the urgent need for innovative approaches to understanding bacterial patho-genesis and developing effective therapies. This review explores the potential of synthetic biology as a promising tool for investigating host-pathogen interactions and offering alternative therapeutic solu-tions for MDR infections. We first examine the progress of CRISPR-based strategies that have enabled modulation of essential gene expression, providing insights into the molecular mechanisms underly-ing host-pathogen interactions. We then discuss the use of engineered microbial synthetic circuits for rapid pathogen detection, which identify molecular signatures involved in interspecies communica-tion and facilitate swift pathogen elimination. Additionally, we explore the potential of Phage Therapy (PT), which leverages bacteriophages to selectively target and eliminate specific bacterial pathogens, presenting a targeted and promising approach to combat MDR infections. Finally, we review the appli-cation of Organ-On-A-Chip (OOAC) technology, which overcomes the limitations of animal models in predicting human immune responses by using microfluidic devices that simulate organ-level physi-ology and pathophysiology, thereby enabling more accurate disease modeling, drug testing, and the development of personalized medicine.

ABSTRACT All great apes differ karyotypically from humans due to the fusion of chromosomes 2a and 2b, resulting in human chromosome 2. Yet, the structure, function, and evolutionary history of the genomic regions associated with this fusion remain poorly understood. Here, we analyze finished telomere-to-telomere chromosomes in great apes and macaques to show that the fusion was associated with multiple pericentric inversions, segmental duplications (SDs), and the rapid turnover of subterminal repetitive DNA. We characterized the fusion site at single-base-pair resolution and identified three distinct SDs that originated more than 5 million years ago. These three distinct SDs were differentially distributed among African great apes as a result of incomplete lineage sorting (ILS) and lineage-specific duplication. Most conspicuously, one of these SDs shares homology to a hypomethylated SD spacer sequence present in hundreds of copies in the subterminal heterochromatin of chimpanzees and bonobos. The fusion in human was accompanied by a systematic degradation of the three divergent α-satellite arrays representing the ancestral centromere creating five distinct structural haplotypes in humans. CRISPR/Cas9-mediated depletion of the fusion site in human cell lines significantly alters the expression of 108 genes, indicating a potential regulatory consequence to this human-specific karyotypic change.

Abstract Biallelic pathogenic variants in the nebulin ( NEB ) gene lead to the congenital muscle disease nemaline myopathy. In-frame deletion of exon 55 (ΔExon55) is the most common disease-causing variant in NEB . Previously, a mouse model of Neb ΔExon55 was developed; however, it presented an uncharacteristically severe phenotype with a near complete reduction in Neb transcript expression that is not observed in NEB exon 55 patients. We identified by RNA sequencing that the cause of this unexpectedly severe presentation in mice is the generation of a pseudoexon containing two premature termination codons (and promoting nonsense mediated decay) at the Neb exon 55 deletion site. To prove that this is the cause of the loss of Neb transcript, and to generate a more faithful model of the human disease, we used CRISPR gene editing to remove the pseudoexon sequence and replace it with human intron 54 sequence containing a validated cas9 gRNA protospacer. The resulting “hmz” mice have a significant reduction in pseudoexon formation (93.6% reduction), and a re-introduction of stable Neb transcript expression. This new model has the characteristic features of nemaline myopathy at the physiological, histological, and molecular levels. Importantly, unlike the existing exon 55 deletion mice (which die by age 7 days), it survives beyond the first months and exhibits obvious signs of neuromuscular dysfunction. It thus provides a new, robust model for studying pathomechanisms and developing therapies for NEB related nemaline myopathy.

Increased expression of K Ca 3.1 has been found in vascular smooth muscle (SMC), macrophages, and T cells in atherosclerotic lesions from humans and mice. Proliferating SMC cells increase the expression of K Ca 3.1, such that it becomes a dominant K + channel and contributes to SMC cell migration. The efficacy of pharmacological inhibition of K Ca 3.1 in limiting atherosclerosis progression has been demonstrated in mice and pigs, however direct, loss-of-function, i.e. gene silencing, studies are absent. To investigate the role of K Ca 3.1, we used CRISPR/Cas9 to generate K Ca 3.1 -/- Apoe -/- (DKO) mice and assessed lesion development in the brachiocephalic artery (BCA) of DKO versus Apoe -/- mice on a Western diet for 3 months. Notably, the loss of K Ca 3.1 did not affect serum total cholesterol or body weight. In BCAs of DKO mice, lesion size (0.036 mm² vs. 0.118 mm², p<0.05) and relative stenosis (13.9% vs. 43.0%, p<0.05) were reduced by 70% compared to Apoe -/- mice, with no effect on medial or lumen area. Additionally, DKO mice exhibited a significant reduction in macrophage content within atherosclerotic plaques compared to Apoe -/- mice, independent of sex. In vitro migration assays further showed a significant reduction in migration of bone marrow-derived macrophages (BMDMs) from DKO mice compared to those from Apoe -/- mice. Furthermore, in vitro experiments using rat aortic smooth muscle cells (RAOSMCs) revealed significant inhibition of PDGF-BB-induced MCP1/Ccl21 expression upon K Ca 3.1 inhibition, while activation of K Ca 3.1 further enhanced MCP1/Ccl21 expression. Both in vivo and in vitro analyses showed that silencing K Ca 3.1 and sex had no significant effect on the collagen content of plaque. RNAseq analysis of BCA samples from DKO and Apoe -/- mice revealed PPAR-dependent signaling as a potential key mediator of the reduction in atherosclerosis due to K Ca 3.1 silencing. Overall, this study provides the first genetic evidence that K Ca 3.1 is a critical regulator of atherosclerotic lesion development and composition and provides novel mechanistic insight into the link between K Ca 3.1 and atherosclerosis.

SUMMARY Following the development of therapeutic probiotics, there is an emerging demand for constraining engineered microbial activities to ensure biosafety. Many biocontainment studies developed genetic devices that involve cell death and growth inhibition on the engineered microbes, which often create basal levels of cytotoxicity that hamper cell fitness and performance on therapeutic functions; furthermore, these toxic pathways may promote genetic instability that leads to mutations and breakdown of biocontainment circuit. To address this issue, here we explore a circuit design that destroys the engineered genetic materials in a probiotic strain, instead of killing these cells, under non-permissive conditions. Our safeguard circuit involves a two-layered transcriptional regulatory circuit to control the expression of a CRISPR system that targets the engineered genes for degradation. In Escherichia coli Nissle 1917 ( EcN ), the biocontainment system continuously scavenged and destroyed the target until no engineered cellular function could be detected, suggesting this strategy has the potential to avoid escapee formation. Additionally, this safeguard circuit did not affect EcN cell fitness. We further demonstrated that the engineered probiotics inhabited in mouse guts and continued the engineered activities for at least 7 days when the permissive signal was supplied constantly; when the permissive signal was not provided, the engineered activities became undetectable within two days. Together, these studies support that our safeguard design is feasible for synthetic probiotic applications. HIGHLIGHTS Our safeguard system only destroys target genes and does not kill the host microbes It terminated engineered activities in guts in response to a loss of a signal This safeguard allowed synthetic probiotics to inhabit in guts for at least a week Cellobiose has great potential to serve as a continuous genetic signal in guts

ABSTRACT A key feature of cytotoxic CD8 + T cells for eliminating pathogens and malignant cells is their capacity to produce pro-inflammatory cytokines, which includes TNF and IFNγ. Provided that these cytokines are highly toxic, a tight control of their production is imperative. RNA-binding proteins (RBPs) are essential for the fine-tuning of cytokine production. The role of the RBP ZFP36L1 and its sister protein ZFP36L2 herein has been established, however, their relative contribution to cytokine production is not well known. We here compared the effect of ZFP36L1 and ZFP36L2 single and double deficiency in murine effector CD8 + T cells. Whereas single deficient T cells significantly increased cytokine production, double deficiency completely unleashed the cytokine production. Not only the TNF production was substantially prolonged in double-deficient T cells. Also, the production of IFNγ reached unprecedented levels with >90% IFNγ–producing T cells compared to 3% in WT T cells, even after 3 days of continuous activation. This continuous cytokine production by double-deficient T cells was also observed in tumor-infiltrating lymphocytes in vivo, however, with no effect on tumor growth. Rather, ZFP36L1 and ZFP36L2 double deficiency resulted in decreased cell viability, impaired STAT5 signaling, and dysregulated cell cycle progression. In conclusion, while combined deletion in ZFP36L1 and ZFP36L2 can drive continuous cytokine production even under chronic activation, safeguards are in place to counteract such super-cytokine producers.

Background Optogenetic systems use light-responsive proteins to control gene expression with the “flip of a switch”. One such tool is the l ight a ctivated C RISPR e ffector (LACE) system. Its ability to regulate gene expression in a tunable, reversible, and spatially resolved manner makes it attractive for many applications. However, LACE relies on delivery of four separate components on individual plasmids, which can limit its use. Here, we optimize LACE to reduce the number of plasmids needed to deliver all four components. Results The two-plasmid LACE (2pLACE) system combines the four components of the original LACE system into two plasmids. Following construction, the behavior of 2pLACE was rigorously tested using optogenetic control of enhanced green fluorescent protein (eGFP) expression as a reporter. We optimized the ratio of the two plasmids, measured activation as a function of light intensity, and determined the frequency of the light to activate the maximum fluorescence. Overall, the 2pLACE system showed a similar dynamic range, tunability, and activation kinetics as the original four plasmid LACE (4pLACE) system. Interestingly, 2pLACE also had less variability in activation signal compared to 4pLACE. Conclusions This simplified system for optogenetics will be more amenable to biotechnology applications where variability needs to be minimized. By optimizing the LACE system to use fewer plasmids, 2pLACE becomes a flexible tool in multiple research applications.

Glioblastoma multiforme (GBM), a highly aggressive brain tumor, frequently develops resistance to temozolomide (TMZ), the current standard chemotherapy. Our study investigates the potential of combining TMZ with the poly (ADP-ribose) polymerase inhibitor Olaparib (OLA) to overcome TMZ resistance. Using in vitro models, including U251 cell lines and patient-derived GBM primary cultures, we demonstrate that OLA enhances TMZ efficacy by disrupting base excision repair and potentiating DNA damage-induced cytotoxicity. A CRISPR knockout screen identified DNA mismatch repair (MMR) gene deficiencies as key drivers of TMZ resistance, which OLA effectively counteracts. Co-treatment with TMZ and OLA significantly reduced tumor cell viability, even in MMR-deficient and MGMT-expressing contexts, suggesting a synergistic mechanism. Gene expression analysis revealed that the combination therapy impacts cell cycle regulation, stress responses, and extracellular matrix integrity, leading to mitotic catastrophe and apoptosis. These findings propose the TMZ-OLA combination as a promising therapeutic strategy for overcoming chemoresistance in GBM.

Uncovering mechanisms by which sensory systems evolve is critical for understanding how organisms adapt to a novel environment. Astyanax mexicanus is a species of fish with populations of surface fish that inhabit rivers and streams and cavefish that have adapted to life within caves. Cavefish have evolved sensory system changes relative to their surface fish counterparts, providing an opportunity to investigate mechanisms underlying sensory system evolution. Here, we report the role of the gene retinal homeobox 3 ( rx3 ) in cavefish eye evolution. We generated surface fish with putative loss-of-function mutations in the rx3 gene using CRISPR-Cas9 to determine the role of this gene in eye development in this species. These rx3 mutant surface fish fail to develop eyes, demonstrating that rx3 is required for surface fish eye development. Further, rx3 mutant surface fish exhibit altered behaviors relative to wild-type surface fish, suggesting that the loss of eyes impacts sensory-dependent behaviors. Finally, eye development is altered in cave-surface hybrid fish that inherit the mutant allele of rx3 from surface fish relative to siblings that inherit a wild-type surface fish rx3 allele, suggesting that cis-regulatory variation at the rx3 locus contributes to eye size evolution in cavefish. Together, these findings demonstrate that, as in other species, rx3 is required for eye development in A. mexicanus . Moreover, they suggest that variation at the rx3 locus plays a role in the evolved reduction of eye size in cavefish, shedding light on the genetic mechanisms underlying sensory system evolution in response to extreme environmental changes.

ABSTRACT Inorganic nitrogen (N) fertilizer has emerged as one of the key factors driving increased crop yields in the past several decades; however, the overuse of chemical N fertilizer has led to severe ecological and environmental burdens. Understanding how crops respond to N fertilizer has become a central topic in plant science and plant genetics, with the ultimate goal of enhancing N use efficiency (NUE) in crop production. As one of the most essential macronutrients, N significantly influences crop performance across different developmental stages of plant, phenotypic traits result from the accumulative effects of genetic factors, prevailing environmental conditions (specifically N availability), and their complex interactions. To characterize the targeting N-responsiveness and growth trajectory, we employed CRISPR-Cas9 technique to generate sorghum mutants using CRISPR technology. Using a LemnaTec plant imaging system, we obtained time series imagery data from 29 to 130 days after sowing (DAS) for these CRISPR-edited mutants under high N and low N greenhouse conditions. After imagery data analysis, we extracted a number of morphological and greenness index traits as a proxy of plant growth and N responses. Subsequently, we employed two different methods to model the temporal N-responsive traits, allowing us to estimate seven key parameters from the growth curve. Our findings revealed that the wildtype and the edited sorghum lines exhibited differences in N responses for several of the key growth-related parameters. The high-throughput N phenotyping pipeline paves the way for a better understanding of the N responses of edited lines in a dynamic manner and sheds light on further improvements in crop NUE.

Sickle cell disease (SCD) is a genetic anemia caused by the production of an abnormal adult hemoglobin. The clinical severity is lessened by elevated fetal hemoglobin (HbF) production in adulthood. A promising therapy is the transplantation of autologous, hematopoietic stem/progenitor cells (HSPCs) treated with CRISPR/Cas9 to downregulate the HbF repressor BCL11A via generation of double strand breaks (DSBs) in the +58-kb erythroid-specific enhancer. Here, to further enhance HbF production without increasing the mutagenic load, we targeted both +58-kb and +55-kb BCL11A erythroid-specific enhancers using base editors. We systematically dissected DNA motifs recognized by the key transcriptional activators within these regions and identified the critical nucleotides required for activator binding. Multiplex base editing of these residues was efficient and safe and generated no or little DSBs and genomic rearrangements. We observed substantial HbF reactivation, exceeding the levels achieved using the CRISPR/Cas9 nuclease-based strategy, thus efficiently rescuing the sickling phenotype. Multiplex base editing was efficient in long-term repopulating HSPCs and resulted in potent HbF reactivation in vivo . In summary, these results show that multiplex base editing of BCL11A erythroid-specific enhancers is a safe and potent strategy for treating sickle cell disease.

ABSTRACT Background Epigenetic regulator genes play critical roles in controlling cell identity and are frequently disrupted in breast cancers, suggesting a key driver role in this disease and its associated phenotypes. However, specific epigenetic drivers (epidrivers) of mammary cell plasticity and their mechanistic contributions to this phenotype are poorly characterized. Methods To identify potential epidrivers of the emergence of mesenchymal breast cancer stem cell-like phenotypes in non-tumorigenic mammary cells, we employed a CRISPR/Cas9 loss-of-function screening strategy targeting epigenetic regulator genes. This approach was followed by an in-depth validation and characterization of epigenomic, transcriptomic, proteomic and phenotypic changes resulting from the disruption of the putative epidriver gene BAP1 . Results Our investigation revealed that loss of the histone deubiquitinase BAP1 impacts cellular processes associated with breast cancer cell plasticity such as epithelial-to-mesenchymal transition (EMT) and actin cytoskeleton organization. In addition, we unveiled that BAP1 loss resulted in an overall less permissive chromatin and downregulated gene expression, impacting programs that control cellular glycosylation and leading to decreased glycan abundance and complexity. BAP1 rescue restored the expression of several deregulated genes in a catalytic activity-dependent manner, suggesting that BAP1-mediated cell identity and glycosylation regulation are largely dependent on its histone deubiquitinase activity. Conclusions Overall, our results point to BAP1 disruption as a driver of mammary cell plasticity and reveal a novel role of BAP1 as an epigenetic regulator of cellular glycosylation.

Dental caries, a prevalent global health issue, results from complex bacterial interactions. In response to harmful stimuli, a desirable outcome for the tooth is the formation of tertiary dentin, a protective reparative process that generates new hard tissue. This reparative dentinogenesis is associated with significant inflammation, which triggers the recruitment and differentiation of dental pulp stem cells (DPSCs). Previously, we have demonstrated that brain-derived neurotrophic factor (BDNF) and its receptor TrkB, key mediators of neural functions, are activated during the DPSC-mediated dentin regeneration process. In this study, we further define the role of inflammation in this process and apply stem cell engineering to enhance dentin regeneration in injured teeth. Our data show that TrkB expression and activation in DPSCs rapidly increase during odontogenic differentiation, further amplified by inflammatory inducers and mediators such as TNFα, LTA, and LPS. An in vivo dentin formation assessment was conducted using a mouse pulp-capping/caries model, where CRISPR-engineered DPSCs overexpressing BDNF were transplanted into inflamed pulp tissue. This transplantation significantly enhanced dentin regeneration in injured teeth. To further explore potential downstream pathways, we conducted transcriptomic profiling of TNFα-treated DPSCs, both with and without TrkB antagonist CTX-B. The results revealed significant changes in gene expression related to immune response, cytokine signaling, and extracellular matrix interactions. Taken together, our study advances our understanding of the role of BDNF in dental tissue engineering using DPSCs and identifies potential therapeutic avenues for improving dental tissue repair and regeneration strategies.

Abstract The neuromuscular junction (NMJ) is the unique interface between lower motor neurons and skeletal muscle fibers and is indispensable for muscle function. Tight control of its localized formation at the center of every muscle fiber, and maintenance throughout lifetime are sustained by muscle-specific kinase (MuSK). MuSK acts as central regulator of acetylcholine receptor clustering at the postsynapse. Localized and temporally controlled signaling of MuSK is primarily achieved by tyrosine autophosphorylation and inhibition thereof. Previous research suggested serine phosphorylation of the activation domain as additional modulator of MuSK activation. Here we identified calcium/calmodulin dependent protein kinase II (CaMK2) and in particular CaMK2β as novel catalyst of MuSK activation and confirmed its capability to phosphorylate MuSK in heterologous cells. However, whereas CaMK2β absence in muscle cells reduced AChR clustering, MuSK phosphorylation was unchanged. Accordingly, we ruled out MuSK phosphorylation as the cause of synapse fragmentation in a mouse model for myotonic dystrophy type 1, in which the muscle-specific splice-variant of CaMK2β is missing, or as the cause of ataxia or delayed muscle development in CaMK2β knockout animals. Histological characterization of muscles of CaMK2β knockout mice indicated specific roles of CaMK2β in fast glycolytic versus slow oxidative muscle. Taken together our data shows that MuSK can be phosphorylated by CaMK2b, but loss of CaMK2b is likely compensated for by other CaMK2 paralogs at the NMJ.

Chronic antigenic stimulation is central to marginal zone lymphoma (MZL) development. While the pharmacological inhibition of the B-cell receptor (BCR) signaling is initially effective, secondary resistance frequently develops. We conducted a CRISPR interference (CRISPRi) screen investigating enhancer-associated long non-coding RNAs (elncRNAs) in MZL cells to identify transcripts crucial to shape their dependence on BCR pathway activation. We identified LOC730338, an elncRNA associated with A-to-I RNA editing, which we renamed ADARreg. Silencing ADARreg re-sensitized tumor cells to BCR pathway inhibition by modulating ADAR2 nuclear translocation and altering intronic RNA editing. The process preferentially affected genes that enhance immune responses, such as STING, IRF3, and p65. ADARreg knockdown also increased lymphoma cell sensitivity to NK cell-mediated cytotoxicity. Our results indicate that targeting elncRNAs like ADARreg represents a potential strategy to overcome drug resistance in lymphoma, also opening new therapeutic opportunities for immunotherapy.

Summary Potato ( Solanum tuberosum ) is the third most important food crop in the world. Although the potato genome has been fully sequenced, functional genomics research of potato lags relative to other major food crops due primarily to the lack of a model experimental potato line. Here, we present a diploid potato line, ‘Jan’, which possesses all essential characteristics for facile functional genomics studies. Jan has a high level of homozygosity after seven generations of self-pollination. Jan is vigorous and highly fertile with outstanding tuber traits, high regeneration rates, and excellent transformation efficiencies. We generated a chromosome-scale genome assembly for Jan, annotated genes, and identified syntelogs relative to the potato reference genome assembly DMv6.1 to facilitate functional genomics. To miniaturize plant architecture, we developed two “mini-Jan” lines with compact and dwarf plant stature using CRISPR/Cas9-mediated mutagenesis targeting the Dwarf and Erecta genes related to growth. Mini-Jan mutants are fully fertile and will permit higher-throughput studies in limited growth chamber and greenhouse space. Thus, Jan and mini-Jan provide an outstanding model system that can be leveraged for gene editing and functional genomics research in potato.

Genetic alterations alone cannot account for the diverse phenotypes of cancer cells. Even cancers with the same driver mutation show significant transcriptional heterogeneity and varied responses to therapy. However, the mechanisms underpinning this heterogeneity remain under-explored. Here, we find that novel enhancer usage is a common feature in acute lymphoblastic leukemia (ALL). In particular, KMT2A::AFF1 ALL, an aggressive leukemia with a poor prognosis and a low mutational burden, exhibits substantial transcriptional heterogeneity between individuals. Using single cell multiome analysis and extensive chromatin profiling, we reveal that much transcriptional heterogeneity in KMT2A::AFF1 ALL is driven by novel enhancer usage. Using high resolution Micro-Capture-C in primary patient samples, we also identify patient-specific enhancer activity at key oncogenes such as MEIS1 and RUNX2 , driving high levels of expression of both oncogenes in a patient-specific manner. Overall, our data show that enhancer heterogeneity is highly prevalent in KMT2A::AFF1 ALL and may also be a mechanism that drives transcriptional heterogeneity in cancer more generally. Key Points Leukemia patients with the same driver mutations often display gene expression differences Using chromatin profiling and high resolution 3C methods we show that enhancer heterogeneity drives gene expression differences

Summary Lysosomes are multifunctional organelles that play important roles in cellular recycling, signaling, and homeostasis, relying on precise trafficking and activation of lysosomal enzymes. While the Golgi apparatus plays a central role in lysosomal enzyme sorting, the mechanisms linking Golgi function to lysosomal activity remain incompletely understood. Here, we identify the Golgi-resident protein GRASP55, but not its paralog GRASP65, as a key regulator of lysosome function. More specifically, we demonstrate that loss of GRASP55 expression leads to missorting and secretion of lysosomal enzymes, lysosomal dysfunction and bloating. GRASP55 deficiency also disrupts lysosomal mTORC1 signaling, reducing the phosphorylation of its lysosomal substrates, TFEB and TFE3, while sparing its non-lysosomal targets. Mechanistically, GRASP55 interacts with GNPTAB, a critical enzyme required for mannose 6-phosphate (M6P) tagging of lysosomal enzymes, and is necessary for its correct trafficking and stability. These findings reveal an essential role for GRASP55 in Golgi-lysosome communication and lysosomal enzyme trafficking, and suggest that GRASP55/GORASP2 may act as a susceptibility gene for lysosomal storage disorder (LSD)-like conditions. Overall, this work underscores the importance of Golgi-mediated protein sorting in lysosome function and lysosomal mTORC1 signaling, and provides insights into the molecular basis of LSD-related pathologies.

Yield improvement is one of the most concerning areas of the “Queen of Oilseed” sesame ( Sesamum indicum L.) for its successful commercialisation. Heterosis breeding is an alternate approach for improving sesame compared to the time- and labour-consuming conventional breeding. However, tedious hand emasculation and pollination processes restrict the implementation of commercial heterosis on sesame. The unavailability of male sterile, restorer, and maintainer lines further complicates the problem. Biotechnological gene manipulation can silence anther-specific vital gene(s) leading to male sterility. Anther-specific gene study is therefore crucial to reach such a goal. In this study, we have cloned two established anther-specific promoters: sesame SiBGproplus (hereafter GN13, NCBI accession no. KT246471 ) and tobacco TA29 (NCBI accession no. X52283 ) in the plant expression vector pCAMBIA2301 producing respective GN13::GUS and TA29::GUS chimeric vectors. We recovered putatively transgenic T0 sesame lines using conventional Agrobacterium -mediated transformation with 1.76% and 1.71% frequencies, respectively. The T0 lines were segregated at the 3:1 Mendelian segregation ratio for kanamycin resistance and generated T1 transgenic lines by self-fertilisation. Most of the T1 transgenics had a single copy of transgene integration. The histological assay revealed the tapetum-specific GUS expression in the T1 transgenic lines; GUS expression was undetected in other tested plant parts. The results depict successful anther/tapetum-specific expression of two promoters in transgenic sesame lines. In further research, these promoters could be used for anther-specific cytotoxic gene expression, RNAi, or CRISPR-mediated mutational approaches to destroy androecium selectively and exhibit male sterility in sesame, resulting in heterosis-mediated improvement.

In adulthood, the regenerative capacity of the injured brain circuit is poor preventing functional restoration. Rehabilitative physical exercise is a promising approach to overcome such functional impairment and the metabolic sensor AMPK has emerged to be a critical mediator for this. However, the mechanistic understanding of upstream and downstream components of AMPK signalling in the physical exercise-mediated enhancement of axon regeneration is not clear. We combined swimming exercise with laser axotomy of posterior lateral microtubule (PLM) neurons of Caenorhabditis elegans to address this question. We found that direct activation of AMPK through AICAR treatment is sufficient to improve axon regeneration and functional recovery. The PAR-4/Liver kinase B1 (LKB1) acts upstream of AMPK to enhance functional recovery following swimming exercise. Using genetics, tissue-specific RNAi, and AICAR treatment, we found that the transcriptional regulators DAF-16 and MDT-15 act downstream of AMPK in mediating the positive effects of swimming. We found that MDT-15 acts in neuron to mediate the benefit of AMPK activation in axon regeneration, whereas DAF-16 acts both in neuron and muscle to promote regrowth downstream to AMPK. We also showed that swimming exercise induces nuclear localization of DAF-16 in an AMPK-dependent manner. Our results showed that neuronal and non-neuronal arms of AMPK signalling play an integrative role in response to physical exercise to promote functional recovery after axon injury. Significance statement Finding: ways to promote functional recovery after accidental damage to the nervous system has been challenging as adult neurons lose the capability to regenerate. Rehabilitation therapy is the most promising approach to improve the health condition of patients with nervous system injury. Even in the roundworm C. elegans , axon regeneration could be enhanced through swimming exercise, which is mediated by the metabolic energy sensor AMP Kinase. In this study, using sensory neurons in worm, we found that PAR-4/ Liver kinase B1 acts upstream of AMPK. Whereas, the transcription factor DAF-16/ FOXO and the transcriptional co-regulator MDT-15 act as downstream signalling arms in muscle and neuron tissues. Excitingly, both of these arms could be harnessed through agonist-mediated activation of AMPK to promote functional recovery in adulthood.

Fibroblasts display complex functions associated with distinct gene expression profiles that influence matrix production and cell communications and the autonomy of tissue development and repair. Thrombospondin-2 (TSP-2), produced by fibroblasts, is a potent angiogenesis inhibitor and negatively associated with tissue repair. Single-cell (sc) sequencing analysis on WT and TSP2KO skin fibroblasts demonstrate distinct cell heterogeneity. Specifically, we found an enrichment of Sox10+ multipotent progenitor cells, identified as Schwann precursor cells, in TSP2KO fibroblasts, while fibrosis-related subpopulations decreased. Immunostaining of tissue and cells validated the increase of this Sox10+ population in KO fibroblasts. Furthermore, in silico analysis suggested enhanced pro-survival signaling, including WNT, TGF-β, and PDGF-β, alongside a reduced BMP4 response. Additionally, the creation of two TSP2KO NIH3T3 cell lines using the CRISPR/Cas9 technique allowed functional and signaling validation in a less complex system. Moreover, KO 3T3 cells exhibited enhanced migration and proliferation, with elevated levels of pro-regenerative molecules including TGF-β3 and Wnt4, and enrichment of nuclear β-catenin. These functional and molecular alterations likely contribute to improved healing and increased neurogenesis in TSP2-deficient wounds. Overall, our findings describe the heterogeneity of dermal fibroblasts and identify pro-regenerative features of TSP2KO fibroblasts.