Abstract Nesfatin-1 is a brain-gut peptide encoded by the nucleobindin-2 (NUCB2) gene. We previously demonstrated that a reduced level of nesfatin-1 in the cerebrospinal fluid, induced by intracerebroventricular injection of a nesfatin-1 antibody, is associated with degeneration of the nigrostriatal dopaminergic system. In combination with evidence that nesfatin-1 mediated the rescue of toxicant induced dopaminergic (DAergic) neuron loss in the substantia nigra (SN), as well as reduced nesfatin-1 levels in the blood of patients with Parkinson’s disease (PD), we raise the hypothesis that nesfatin-1 may be essential for the survival of DAergic neurons in SN in mice. In the present study, we found that whole-body Nucb2 knockout via CRISPR/Cas9 technology in mice led to nigrostriatal dopaminergic system degeneration, as evidenced by a reduction in tyrosine hydrolyses-immunoreactivity neurons in the SN, decreased levels of dopamine and its metabolites in the striatum, and mitochondrial and nuclear impairment in the SN. The underlying mechanism may involve oxidative stress and neuroinflammation induced by down-regulation of circadian rhythm-related gene expression. Furthermore, Nucb2 deletion in mice leads to intestinal microecological imbalance, disorder of the bacterial community structure, metabolic homeostasis disruption, and decreased abundance of some sleep rhythm-related bacterial communities and metabolites. Our findings reported that nesfatin-1 plays a role in maintaining the normal function of the nigrostriatal dopaminergic system, which may provide new therapeutic targets for PD.

Abstract Background Over 300 mutations in PSEN1 have been identified as causes of early-onset Alzheimer’s disease (EOAD). While these include missense mutations and a few insertions, deletions, or duplications, none result in open reading frame shifts, and all alter γ-secretase function to increase the long/short Aβ ratio. Methods We identified a novel heterozygous PSEN1 nonsense variant, c.325A > T, in a patient and his father, both presenting with EOAD, resulting in the substitution of lysine 109 with a premature stop codon at position (p.K109*). This produces a truncated 109 amino acid (aa) N-terminal PSEN1 fragment. Functional characterization was performed using overexpression models and a heterozygous mouse model (Psen1 K109*/+ ). Results In overexpression models, downstream ATGs serve as alternative starting codons, generating a > 37kDa and a > 27 kDa PSEN1 C-terminal fragment (PSEN1-CTF A and PSEN1-CTF B , respectively) that retain the two catalytic aspartates of γ-secretase. Heterozygous Psen1 K109*/+ mice exhibited subtle phenotypic defects, including reduced Pen2 expression and mild APP-CTF accumulation. Notably, aged mice demonstrated significantly increased Psen2 protein expression, potentially contributing to an elevated Aβ42/Aβ38 ratio. Conclusions These findings indicate that PSEN1 c.325A > T (p.K109*) is not a complete loss-of-function mutation. However, to what extent and by what mechanism it contributes to EOAD pathogenesis remains unclear.

Bowman-Birk inhibitors (BBI) are an ancient class of serine protease inhibitors originating prior to the emergence of the angiosperms. While BBIs have been preserved in the legume (Fabaceae) and cereal (Poaceae) families, they have been lost in many other divergent lineages. However, their underlying molecular evolution and regulation of BBI remain largely uncharacterized. Our study shows that BBIs in legumes and cereals are encoded by two large and divergent gene families. BBI genes in legumes have further diversified into two subfamilies with distinct gene expression patterns. Genes in one legume BBI subfamily are specifically expressed in seeds while BBI genes in the other legume subfamily and cereal do not have significant expressions in any examined tissues including seed, root, leaf and flower. The soybean BBI gene family shows evidence of expansion via whole genome, segmental and tandem duplication. Protein sequence and structural analysis predicts that functional domains for double-headed inhibitory loops and binding abilities to trypsin and chymotrypsin are largely preserved within the soybean BBI family. The seed-specific subfamily genes are specifically expressed at maturation stages and not at embryogenesis stages. The other, non-seed BBI subfamily genes are highly responsive to a distinct spectrum of signals related to abiotic and biotic stresses. Their specific expression under non-essential biological processes for plant growth and development suggests that, although BBIs have been retained in both cereals and legumes, likely due to their role in enhancing plant fitness under natural selection pressures, they are not involved in core developmental processes. This may explain why BBIs were lost in many divergent plant lineages and support their well-established roles in plant adaptation to environmental stress. Having knocked out the seed-specific BBIs through a CRISPR/Cas9 approach, we have successfully generated soybeans which exhibited 69.4 - 73.7% reduction of trypsin inhibitor activity and 76.4 - 79.4% reduced chymotrypsin inhibitor activity. The edited soybean did not show significant changes in key agronomic traits, supporting that the functions of BBIs are not essential. While BBIs in soybean seeds may have a desirable function in natural selection, they are antinutrients from an applied perspective for their use in feed and food. It provides an opportunity to reduce BBIs in seeds for quality improvement. Our findings provide insights into molecular evolution, regulation, and function of BBI in plants, and successfully demonstrate engineering BBI in seeds to result in production of food and feed of higher nutritional value with minimal impacts on the agronomic performance of the plant.

There is a continued need for identification of novel disease drivers of acute myeloid leukemia as many patients experience relapse and have poor clinical outcomes. Analyses from our study and publicly available datasets predicted CEBPD as a novel tumor suppressor gene in acute myeloid leukemia. Consistent with the analyses, CEBPD knockdown experiments showed activation of MAPK signaling with concomitant increase in cell growth rate, while upregulation experiments suggested induction of myeloid differentiation marker CD14 expression in AML cell lines OCI-AML2 and OCI-AML5. Consistent with a previous report, our genomics analyses and azacytidine treatment experiments suggested a role for DNA methylation in downregulation of CEBPD expression during AML pathogenesis. Altogether, our results provide experimental evidence for a tumor suppressor function of CEBPD in AML.

3-Hydroxypropionic acid (3-HP) is a platform compound that can produce many chemical commodities. This study focuses on establishing and optimizing the production of 3-HP in E. coli . We constructed a series of engineered E. coli strains which can produce 3-HP via the malonyl-CoA pathway. To increase the metabolic flux of acetyl-CoA, a precursor for the synthesis of 3-HP, CRISPR/Cas9-based DNA editing technique was used to knock out the genes encoding pyruvate oxidase ( poxB) , lactate dehydrogenase ( ldhA ) and phosphate transacetylase ( pta ), thereby reducing the formation of by-products. Concurrently, the acetyl coenzyme a carboxylase gene ( accDABC ) is overexpressed on the chromosome with the objective of augmenting intracellular acetyl-CoA levels and, consequently, 3-HP production. Next, we introduced a plasmid containing a codon-optimized malonyl-CoA reductase gene ( mcr ) into the engineered strain. Finally, we constructed a transcription factor-based metabolite biosensor utilizing the PpHpdR/P hpdH system, followed by the screening of mutant strains for enhanced 3-HP production through adaptive laboratory evolution. Combining the above metabolic engineering efforts with optimisation of media and fermentation conditions, the 3-HP titer of the engineered strain WY7 increased from an initial titer 0.34 g/L to 48.8 g/L. This study encourages further research in metabolic pathway optimizationto produce 3-HP. Highlights Synthesis 3-HP in the malonyl-CoA pathway. Edit the Escherichia coli genome using the CRISPR/Cas9 system. Elevated production of 3-HP by knocking out bypass genes ldhA / pta / poxB . A biosensor was designed to respond to 3-HP concentration. Adaptive laboratory evolutionary strategies increase 3-HP production. Abstract Figure

ABSTRACT The continued development of high-dimensional CRISPR screen readouts, such as single-cell RNA sequencing and high-content imaging, necessitates compact libraries to enable functional interrogation at genome scale. Improved genome annotations yield library deprecation over time, further motivating an updated genome-wide design effort. Recently, we have developed an enhanced model, Rule Set 3, which leveraged an expansive training set and feature space to predict guide efficacy. However, the benefit of such advances to library design is limited by current approaches to balance predictions of on-target activity with off-target considerations. Here we present a guide selection strategy that identifies guides with sufficient off-target activity to justify omission from the library, thus avoiding the unnecessary exclusion of active guides. We pair this model with strategic design choices to create Jacquere, an updated, optimized, and validated Cas9 CRISPR knockout (CRISPRko) genome-wide library for the human genome.

The CRISPR-Cas system serves as an adaptive immune defence in bacteria, protecting against foreign genetic elements. In Acinetobacter baumannii , efflux pumps are major contributors to multidrug resistance (MDR). This study investigates the potential regulatory role of the CRISPR-Cas system on efflux pump genes, specifically adeB , and its association with antibiotic resistance. Methods A total of 100 clinical specimens were collected from patients admitted to the Wound Unit at Al-Hilla Teaching Hospital between March and May 2025. Standard bacteriological methods were used for isolation and identification. Antimicrobial susceptibility testing (AST) was conducted using the disk diffusion technique and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) 2025 guidelines. PCR assays were used to detect the presence of CRISPR-Cas system components and the blaOXA-51 gene. Quantitative real-time PCR (qRT-PCR) was employed to assess the expression levels of the adeB efflux pump gene. Results Out of the 100 clinical samples (44 females and 55 males, aged 10–55 years), 15 (15%) isolates were confirmed as A. baumannii . AST results indicated high resistance rates to oxacillin (100%), benzylpenicillin (93.3%), erythromycin (73.3%), and tetracycline (66.7%). The isolates exhibited the highest sensitivity to tigecycline (93.3%), trimethoprim/sulfamethoxazole (93.3%), and rifampicin (86.7%). All isolates were positive for the blaOXA-51 gene.Molecular analysis revealed that the I-Fb subtype of the cas1 gene was present in 86.7% of the isolates. Expression profiling showed that adeB was overexpressed in 66.6% of the isolates. Notably, isolates harboring complete CRISPR-Cas components exhibited downregulation of adeB , suggesting a possible repressive regulatory effect of CRISPR-Cas on efflux pump expression. Conclusion This study demonstrates variability in the distribution of CRISPR-Cas elements among clinical A. baumannii isolates and suggests a potential inverse correlation between CRISPR-Cas system presence—particularly the I-Fb-cas1 subtype—and adeB efflux pump gene expression. These findings highlight the potential of CRISPR-Cas systems to modulate resistance mechanisms in A. baumannii , warranting further investigation into their therapeutic implications.

Intrinsic innate immune barriers have evolved to suppress viral infection and can reduce effective gene delivery in gene therapy. We have developed BG147, a novel cyclosporine A analogue, optimised via structure-guided design to prevent inhibition of HIV cofactor Cyclophilin A and to specifically inhibit interferon-induced transmembrane proteins (IFITM1-3). BG147 enhances VSV-G pseudotyped lentiviral vector transduction ex vivo in hematopoietic stem and progenitor cells (HSPCs) and in in vivo ocular gene therapy of photoreceptor cells in mice. Upon BG147 treatment, IFITM proteins are mislocalised and degraded through lysosomal acidification-dependent pathways. IFITM3 levels functionally return in cells 96 h after BG147 washout. BG147 promises to transform ex vivo and in vivo eye gene therapies by transiently inhibiting intrinsic immune barriers mediated by IFITM proteins to enhance a wide range of protocols. One Sentence Summary Modified cyclosporine, BG147, enhances lentivector gene therapy transduction, ex vivo in HSPC and in vivo in mouse photoreceptors, by degrading IFITM3.

Group B Streptococcus (GBS), a common colonizer of the human genital and gastrointestinal tracts, is a leading cause of neonatal bacterial meningitis, which can lead to severe neurological complications. The hypervirulent serotype III, sequence type 17 (ST-17) strain COH1 is strongly associated with late-onset disease due to its unique set of virulence factors. However, genetic manipulation of ST-17 strains is notoriously challenging, limiting the ability to study key pathogenic genes. In this study, we developed a CRISPR interference (CRISPRi) system utilizing an endogenous catalytically inactivated Cas9 (dCas9) in the COH1 strain, enabling targeted and tunable gene expression knockdown. We confirmed the efficacy of this system through hemolysis assays, qPCR transcriptional analysis, and in vitro infection models using human brain endothelial cells. The CRISPRi system successfully produced phenotypic knockdowns of essential virulence genes, including pilA, srr2 , and iagA , reducing adhesion, invasion, and inflammatory responses at the blood-brain barrier. This platform enables rapid gene knockdowns for functional genomics in ST-17 GBS, enabling high-throughput screening and pathogenesis research. Importance Group B Streptococcus (GBS) remains the world’s leading cause of neonatal meningitis. GBS-host interactions at the blood-brain barrier (BBB) are dependent on bacterial factors, including surface factors and two-component systems. Multi-locus sequence type 17 (ST-17) GBS strains are highly associated with neonatal meningitis, and these strains harbor many virulence factors for infection at the BBB. Historically, these factors have been studied using traditional knockout mutagenesis, which has proven challenging in the most common ST-17 lab strain, COH1. This study utilizes CRISPR interference (CRISPRi) to generate rapid expression knockdown. This study validates a CRISPRi-enabled COH1 dCas9 strain as a versatile tool for probing GBS pathogenesis at the BBB.

Programmable DNA integration using CRISPR-associated transposons (CASTs) offers powerful capabilities for genome engineering. The single effector Cas12k CAST examples evolved from a fixed guide TnpB nuclease protein. Here, we engineer de novo RNA-guided transposition systems, where the single guide RNA effector components are repurposed nuclease-dead TnpB-family proteins. These compact systems mediate high-efficiency guide RNA-directed DNA insertion with preserved orientation control and target immunity, reduced off-site targeting, release of a host factor requirement, and can be paired with an exonuclease domain to mediate cut-and-paste transposition. In this engineered context, the TnpB derivatives show features not predicted from the original enzymes suggesting untapped avenues for improvement. In parallel, we show that mutations at the TniQ-TnsC interface in the Cas12k CAST system selectively attenuate off-site insertions while enhancing on-site activity. These results establish how Cas12 proteins and antecedent TnpB proteins can be engineered for high performance and specificity with guide RNA directed systems.

Loeys-Dietz Syndrome type 3 (LDS3) is caused by pathogenic (P)/likely pathogenic (LP) variants in the SMAD3 gene and is characterized by aneurysm formation and arterial tortuosity, which can lead to life-threatening complications. There is an unmet need for suitable cell models to study LDS3 at a cellular and molecular level. Induced pluripotent stem (iPS) cells offer a promising approach because they can be genetically modified using CRISPR/Cas9 technology and differentiated into disease-relevant cell types. As it is difficult to obtain aortic vascular smooth muscle cells (VSMCs) from patients, iPS cells differentiated into VSMCs provide an ideal model to study cellular aneurysmal phenotypes. In this study, we generated iPS cell models carrying (P/LP) SMAD3 variants. These cell models were generated either by using CRISPR/Cas9 mediated introduction of indels and deletions to introduce SMAD3 variants, or by reprogramming of fibroblasts derived from SMAD3 patients. These iPS cell lines were characterized for SMAD3 expression by Western blotting and validated for pluripotency through immunofluorescence and qPCR. Moreover, the patient-derived iPS cell lines were shown to differentiate into smooth muscle cells (SMCs), which are relevant to study the molecular mechanisms underlying aneurysm formation in LDS3 patients. Our findings highlight the potential of these iPS-based models to investigate the pathophysiology of LDS3 and facilitate the development of therapeutic strategies for aortic aneurysms.

Heterochromatin proteins play a key role in establishing local chromatin structure to control the transcription of target genes. Here we uncover a surprising segregation between regions of high DNA- and high heterochromatin protein 1 β (HP1 β )-density in mouse ES cells. DNA-low/HP1 β -high foci retain freely diffusing HP1 β , and form via condensation through a multitude of weak interactions on top of HP1 β that is bound stably to chromatin. DNA-high/HP1 β -low foci exclude freely diffusing HP1 β and display reduced chromatin mobility, suggesting a higher degree of chromatin self-interaction and a more repressive environment. Finally, the two types of environments are intertwined in DNA-high/HP1 β -high foci, where HP1 β maintains heterochromatin in a more compact yet dynamic chromatin state. During the exit from naïve pluripotency HP1 β is lost from regions of high DNA density as cells transition through the formative state, which might facilitate the reconfiguration of genome structure accompanying a change in cell state that we observed previously. Subsequently, as cells enter primed pluripotency, canonical heterochromatin is established.

Abstract Herein, we investigated the role of an essential transcription factor in the human T-cell leukemia virus type 1 (HTLV-1) provirus, the HTLV-1 basic zip factor (HBZ), in HTLV-1 infections and adult T-cell leukemia/lymphoma (ATL). We designed five synthetic guide RNAs (sgRNAs) targeting HBZ and introduced them into ATL and HTLV-1 infected cell lines using clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Of all sgRNAs, sgRNA 171 was the most efficient in introducing mutations at the target site as 70–80% of Cas9/sgRNA 171-transfected host cells contained mutations. Various types of mutations, including deletions, substitutions, insertion, and combinations, were detected in the Cas9/sgRNA 171-treated cells. Based on the predicted peptide sequence, most mutant clones were assumed to inactivate the HBZ mRNA. The mRNA levels of the transactivator from the X-gene region ( tax ) increased after HBZ editing by Cas9/sgRNA 171. No off-target effects were observed in the four human genome regions partially homologous to the sgRNA 171 target sequence. Furthermore, ST-1 cells transfected with Cas9/sgRNA 171 displayed significantly reduced proliferation. These findings suggest that the HBZ mRNA might be crucial for the survival of HTLV-1-infected cells, including ATL, providing insights into the molecular pathogenesis of the HTLV-1 provirus.

Abstract Mutations in ABCA4 gene causes Stargardt macular degeneration, which manifests with toxic lipofuscin deposits in the outer retina, gradual atrophy of RPE cells, followed by photoreceptor cell loss. The cone-enriched retina, with macula-like ‘area-temporalis’ of zebrafish are better models than rodents for studying human macular dystrophies. Here, we generated abca4b knockout zebrafish model using CRISPR/Cas9 editing and evaluated the early and late-stage retinal changes. In adult abca4b −/− mutants, the RPE cells exhibited hyperpigmentation, altered retinomotor behaviour and lipofuscin accumulation, but they remained viable. However, the photoreceptors underwent progressive degeneration, with a sequential loss of blue and UV cones, followed by red and green cones and finally the rod cells. This triggered the chronic activation and early depletion of retinal stem cells at the ciliary marginal zone of mutants and resulted in accelerated outer-retinal degeneration and severe visual defects, despite them retaining the Müller glia-dependant retinal repair potential.

ABSTRACT Marek’s disease (MD), a highly contagious avian immunosuppressive disorder caused by the α-herpesvirus MDV-1, poses a significant threat to poultry health. The development of rapid visual detection methods capable of distinguishing epidemic MDV-1 strains from vaccine strains is crucial for early disease warning, vaccine efficacy evaluation, and precise disease control. We developed a novel isothermal detection system that integrates recombinase polymerase amplification (RPA) with CRISPR/Cas14a technology for the visual identification of epidemic MDV-1 strains. This method operates at a constant temperature of 37°C and allows for either real-time analysis or endpoint visual readout without the need for complex instrumentation. Our results showed no cross-reactivity with Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), MDV-1 vaccine strains, or herpesvirus of turkeys (HVT). Plasmid DNA standards were used to determine the sensitivity of the assay and the detection limit was 24.6 copies/μL. Clinical evaluation using 24 field samples confirmed that the method successfully identified all MDV-positive cases, demonstrating its diagnostic reliability. In conclusion, we have developed a rapid, instrument-free, and highly specific nucleic acid detection platform for MDV-1 by combining the sensitivity of RPA with the specificity of CRISPR/Cas14a technology, offering promising potential for field-based diagnostics and disease surveillance. IMPORTANCE Marek’s disease virus (MDV-1) is a highly contagious and economically important avian pathogen. Existing diagnostic methods are unable to reliably distinguish between epidemic and vaccine strains in field settings, which hampers effective surveillance and evaluation of vaccination programs. To address this challenge, we developed a portable isothermal detection assay that combines recombinase polymerase amplification (RPA) with CRISPR/Cas14a technology. This approach enables highly sensitive (24.6 copies/μL) and specific visual detection of epidemic MDV-1 strains without cross-reactivity with vaccine strains or related viruses. The assay demonstrated 100% agreement with reference methods when validated using clinical samples. As a cost-effective and instrument-free method, it offers a practical solution for rapid on-site diagnosis, facilitating enhanced outbreak control and improved poultry health management globally.

Avian influenza viruses (AIVs) are zoonotic pathogens that pose an increasing global threat due to their potential for significant economic losses in agriculture, spillover into humans, and the risk of a pandemic should human-to-human transmission occur. These concerns underscore the need for rapid, sensitive and specific tools to detect and differentiate circulating AIV subtypes and clades. Current AIV diagnostic methods rely on specialized equipment and trained personnel, limiting their use in the field and in low-resource settings. Here, we extended SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a CRISPR-based platform, to detect and subtype AIVs. We designed, optimized, and validated SHINE assay for the H5 AIV detection using both fluorescence and lateral flow readout, achieving 100% specificity with PCR-based assays when tested on seasonal influenza-positive clinical samples, and a limit of detection of 121.7 copies/μL on vaccine-derived H5 viral seedstocks. To expand the scope of avian influenza detection, we also designed and validated a SHINE assay targeting the 2.3.4.4b A(H5N1) lineage, in response to the ongoing H5N1 outbreak in cattle in the United States, and a SHINE assay specific to Eurasian H7 lineage to discriminate against North American H7 lineage. Together, these SHINE assays offer a promising platform for AIV diagnosis and surveillance, particularly in settings with limited laboratory infrastructure.

Neurological disorders often originate from progressive brain network dysfunctions that start years before symptoms appear. How these changes emerge in the developing human brain remains elusive due to a lack of tractable model systems. Here, we show a cerebral organoid model of Tuberous Sclerosis Complex (TSC) that recapitulates hallmarks of epileptogenesis in vitro. We compare extracellular recordings of TSC organoids with intraoperative electrocorticography from TSC patients to reveal striking functional similarities, including high-frequency oscillations - an electrical biomarker for epileptogenic tissue. In TSC, a human-specific interneuron sub-type derived from the caudal ganglionic eminence drives network hyper-synchronization through increased spontaneous firing and altered excitability. Inhibiting overproliferation of its progenitors via long-term epidermal growth factor receptor inhibition prevented the onset of this pathological phenotype at functional and morphological levels. Our work shows that organoids allow mechanistic analysis of emerging neural network phenotypes, enabling anti-epileptogenic drug testing in a human brain development model.

Ovarian high-grade serous cancer (HGSC) is an aggressive subtype of epithelial ovarian cancer. Here, we identify BX-912, a phosphoinositide-dependent kinase 1 (PDPK1) inhibitor, as a promising therapeutic agent for HGSC. BX-912 suppressed HGSC growth as a single agent and synergized with olaparib independently of BRCA status. Unexpectedly, BX-912 treatment induced multinucleation, a phenotype not observed with other PDPK1 inhibitors. Proteome Integral Solubility Alteration (PISA) profiling revealed the transcription factor HES1 as a functional target of BX-912. Structural modeling showed that BX-912 binds the Orange domain of HES1, while its WRPW motif mediates interactions with protein partners, including the AP2 endocytic protein complex, coordinating their nuclear accumulation that leads to a mitotic catastrophe. Furthermore, cell cycle analyses showed that BX-912 combined with olaparib synergistically enhanced DNA damage and G2-M arrest. Our study demonstrates the value of proteomics for revealing hidden drug activities. It also identifies potential inhibition strategies for HES1, which is commonly overexpressed in HGSC. Additionally, this study proposes a novel strategy of targeting consecutive cell cycle phases to enhance treatment efficacy in HGSC.

A key pathological feature of Parkinsons Disease (PD) is the loss of neuromelanin, an iron chelator within the dopaminergic neurons, which results in iron toxicity thought to result in selective neuronal vulnerability. This implicated iron handling pathways as an early target of research in PD. Given the critical role of PD-related activating mutations in LRRK2 (leucine-rich repeat protein kinase 2) within membrane trafficking pathways we examined the impact of mutant LRRK2G2019S on iron homeostasis within a model macrophage cell line known to have high iron capacity. Proteomics analysis revealed a dysregulation of iron-related proteins in steady state with highly elevated levels of ferritin light chain and a reduction of ferritin heavy chain. LRRK2 mutant cells showed efficient ferritinophagy upon iron chelation, but upon iron overload there was a near complete block in the degradation of the ferritinophagy adaptor NCOA4. Surprisingly, NCOA4 levels were not rescued upon inhibition of the LRRK2 kinase activity in iron overload conditions, nor was the phosphorylation of substrate Rab GTPases. We therefore generated a CRISPR mutation to delete the kinase domain of LRRK2 and express only the Rab-binding armadillo repeat domain. Although the kinase domain was deleted, the truncation mutant of LRRK2 showed strong Rab8 phosphorylation in conditions of iron overload, similar to LRRK2G2019S cells, with the phosphorylated Rab8 accumulating at the plasma membrane. These data indicate that the G2019S mutation acts as a kinase independent, dominant-interfering mutant specifically in conditions of iron overload. Together, our data implicate LRRK2 as a key regulator of iron homeostasis and point to the need for an increased focus on the mechanisms of iron dysregulation in PD.

ABSTRACT A scarcity of live, paralog-specific tools has limited analysis of PSD-MAGUKs at excitatory synapses. To address this gap, we engineered small, 10 FN3-derived binders that selectively recognize PSD-93 and SAP102 -alongside an enhanced PSD-95 reagent- and converted them into regulated, gene-encoded intrabodies for endogenous imaging. Through sequence-guided selection and targeted optimization, we obtained high-specificity reagents that label their native targets in neurons with minimal perturbation and support multiplexed live-cell and advanced imaging modalities. This toolkit enables differential visualization of MAGUK paralogs at native levels and provides a practical route to dissect their distinct contributions to synapse organization and plasticity.

The class 1 HDACs 1, 2 and 3 form seven families of distinct large multiprotein complexes that regulate gene expression via deacetylation of lysines in histone tails. The degree of redundancy and functional overlap between complexes and their primary gene targets, remains unknown. We used CRISPR/Cas9 to independently tag HDAC complexes with FKBP12 F36V in HCT116 cells enabling rapid ( 50% of expressed genes. More than 60% of these are specific to an individual complex. Of genes regulated by more than one complex, approaching 50% are reciprocally regulated such that HDAC complexes act as antagonistic regulators. Homer analysis strongly suggests that the complexes are reliant on different transcription factors. This is the first study to identify the primary targets of individual HDAC complexes and directly compare the effects of rapid degradation on gene regulation in the same biological system.

The diverse pigmentation patterns of animals are crucial for predation avoidance and behavioral display, yet mechanisms underlying this diversity remain poorly understood. In zebrafish, Turing models have been proposed to explain stripe patterns, but it is unclear if they apply to other fishes. In anemonefish ( Amphiprion ocellaris) , we identified gja5b , a gene orthologous to zebrafish leopard and encoding a connexin involved in pigment cell communication, as responsible for the Snowflake phenotype. Using CRISPR/Cas9 and transgenesis, we recapitulate the Snowflake phenotype and show expression of gja5b in iridophores. A matching allele was recovered in zebrafish, revealing complementary requirements in both species. Our findings highlight conserved roles of gap junction mediated communication in pigment patterning across divergent teleosts.

ABSTRACT The persistent residual tumor cells that survive after chemotherapy are a major cause of treatment failure, but their survival mechanisms remain largely elusive. These cancer cells are typically characterized by a quiescent state with suppressed activity of MYC and MTOR. We observed that the MYC-suppressed persistent triple-negative breast cancer (TNBC) cells are metabolically flexible and can upregulate mitochondrial oxidative phosphorylation (OXPHOS) genes and respiratory function (“OXPHOS-high” cell state) in response to DNA-damaging anthracyclines such as doxorubicin, but not to taxanes. The elevated biomass and respiratory function of mitochondria in OXPHOS-high persistent cancer cells were associated with mitochondrial elongation and remodeling suggestive of increased mitochondrial fusion. A genome-wide CRISPR editing screen in doxorubicin-persistent OXPHOS-high TNBC cells revealed BCL-XL gene as the top survival dependency in these quiescent tumor cells, but not in their untreated proliferating counterparts. Quiescent OXPHOS-high TNBC cells were highly sensitive to BCL-XL inhibitors, but not to inhibitors of BCL2 and MCL1. Interestingly, inhibition of BCL-XL in doxorubicin-persistent OXPHOS-high TNBC cells rapidly abrogated mitochondrial elongation and respiratory function, followed by caspase 3/7 activation and cell death. The platelet-sparing proteolysis targeted chimera (PROTAC) BCL-XL degrader DT2216 enhanced the efficacy of doxorubicin against TNBC xenografts in vivo without induction of thrombocytopenia that is often observed with the first-generation BCL-XL inhibitors, supporting the development of this combinatorial treatment strategy for eliminating dormant tumor cells that persist after treatment with anthracycline-based chemotherapy.

Cytotoxic CD8 + T-cells play central roles in tumor immunotherapy. Understanding mechanisms that regulate development, differentiation, and functions of cytotoxic CD8 + T-cells leads to development of better immunotherapies. By combining primary T-cell culture and a syngeneic mouse tumor model with both genome-wide and custom CRISPR/Cas9 screenings, we systematically identified genes and pathways that regulate PD-1 expression and functions of CD8 + T-cells. Among them, inactivation of a key enzyme in glycoconjugate biosynthesis, beta 1, 4-galatosyltransferase 1 (B4GALT1), leads to significantly enhanced T-cell receptor (TCR) activation and functions of CD8 + T-cell. Interestingly, suppression of B4GALT1 enhances functions of TCR-T-cells, but has no effect on chimeric antigen receptor T (CAR-T) cells. We systematically identified the substrates of B4GALT1 on CD8 + T-cell surface by affinity purification and mass spectrometry analysis, which include protein components in both TCR and its co-receptor complexes. The galactosylation of TCR and CD8 leads to reduced interaction between TCR and CD8 that is essential for TCR activation. Artificially tethering TCR and CD8 by a TCR-CD8 fusion protein could bypass the regulation of B4GALT1 in CD8 + T-cells. Finally, the expression levels of B4GALT1 normalized to tumor infiltrated CD8 + T-cells in tumor microenvironment are significant and negatively associated with prognosis of human patients. Our results reveal the important roles of protein N-glycosylation in regulating functions of CD8 + T-cells and prove that B4GALT1 is a potential target for tumor immunotherapy.

Although glucocorticoids are widely used to alleviate side effects of prostate cancer (PCa) treatment, the glucocorticoid receptor (GR) exhibits a dual role exerting tumor-suppressive effects by inhibiting early-stage PCa cell proliferation, while also promoting oncogenic progression by mediating antiandrogen resistance. The mechanisms underlying this functional dichotomy have remained elusive and poorly characterized. Using genome-wide analyses and CRISPR-based genome editing, we identified the tumor protein p63 as a key mediator of GR’s tumor-suppressive chromatin activity. Loss of p63 reprograms GR activity toward an oncogenic state, marked by enhanced cell migration, invasion, and altered morphology. This shift is driven by increased GATA2 expression, which alters GR’s chromatin binding and transcriptional output. Together, our findings uncover a p63–GATA2 molecular switch that governs the dual role of GR in PCa, establishing transcription factor crosstalk as a critical regulator of GR-driven oncogenic reprogramming and cellular plasticity.