Summary Microglia, the brain’s innate immune cells, can adopt a wide variety of activation states relevant to health and disease. Dysregulation of microglial activation occurs in numerous brain disorders, and driving or inhibiting specific states could be therapeutic. To discover regulators of microglial activation states, we conducted CRISPR interference screens in iPSC-derived microglia for inhibitors and activators of six microglial states. We identified transcriptional regulators for each of these states and characterized 31 regulators at the single-cell transcriptomic and cell-surface proteome level in two distinct iPSC-derived microglia models. Finally, we functionally characterized several regulators. STAT2 knockdown inhibits interferon response and lysosomal function. PRDM1 knockdown drives disease-associated and lipid-rich signatures and enhanced phagocytosis. DNMT1 knockdown results in widespread loss of methylation, activating negative regulators of interferon signaling. These findings provide a framework to direct microglial activation to selectively enrich microglial activation states, define their functional outputs, and inform future therapies. Highlights CRISPRi screening reveals novel regulators of six microglia activation states Multi-modal single-cell screens highlight differences between mRNA and protein level expression iPSC-microglia models show different baseline distributions of activation states Loss of DNMT1 leads to widespread DNA demethylation, promoting some states but limiting the interferon-response state Loss of PRDM1 drives microglial disease-associated state

In early 2025, Sierra Leone experienced a large outbreak of mpox clade IIb, underscoring the urgent need for portable, low-cost diagnostics in decentralized settings. We rapidly developed and field-deployed Mpox SHINE, a CRISPR–Cas13 assay that integrates lyophilized reagents, ambient-temperature lysis, and automated fluorescence detection on a portable device, the DxHub. The assay achieved analytical sensitivity down to 10 copies/µL with minimal hands-on time. In-country evaluation of 56 clinical specimens showed complete concordance with qPCR, with 100% sensitivity (45/45) and 100% specificity (11/11) at the sample level. Mpox SHINE also detected the virus directly from unextracted lesion swabs, maintaining 100% sensitivity and specificity in 16 samples (8 positive, 8 negative). Across extracted and unextracted samples, the mean time-to-result was ∼35 minutes, with all positives detected within 45 minutes. Thus, Mpox SHINE performed on the DxHub demonstrates how CRISPR-based pathogen detection can be rapidly translated into portable tools for the front lines of outbreak response. Teaser CRISPR-Cas13 assay and portable device bring rapid, reliable mpox testing to outbreak settings.

X-linked Dystonia-Parkinsonism (XDP) is a lethal adult-onset neurodegenerative disorder that exhibits features of dystonia and parkinsonism and is exclusively associated with a causal founder haplotype that is indigenous to the Philippines and affects Filipino males. Using patient-specific fibroblasts, neural stem cells (NSC), and other neuronal models, we discovered that cryptic alternative splicing caused by a novel SINE-VNTR-Alu (SVA) mobile element insertion into intron 32 of TAF1 is a mechanistic hallmark of XDP. We leveraged postmortem brain samples from an XDP-specific brain bank to demonstrate that the molecular hallmarks of XDP observed in neural stem cells (NSCs) mirror abnormalities observed in brain tissues from affected patients. Based on these findings that patient-specific NSCs reproduce mechanistic signatures found in the brain, we sought to develop a bespoke precision therapeutic for XDP and evaluate its relative efficacy in ameliorating transcriptomic signatures in neuronal models. We first used CRISPR-based excision of the SVA and demonstrated ablation of all aberrant splicing and dysregulation of TAF1 expression in NSCs across 30 independent clones. CRISPR-based correction of the XDP haplotype also restored the expression of 424 of 1,490 (30%) differentially expressed genes (DEGs) that were altered in XDP patient lines and greatly exceeded what would be expected by chance (p-value = 9.89e-87). While in vivo delivery of a gold standard CRISPR therapy is currently not feasible for XDP, we evaluated a tractable approach for Filipino patients by exploring the potential to modulate alternative splicing in XDP patients using antisense oligonucleotides (ASOs). To accomplish this, we developed a large-scale and well controlled functional genomics platform that screened eighty ASOs targeting intron 32 of XDP patients, followed by prioritization of lead ASOs based on attenuation of the alternative splicing signature. In transcriptome analyses across 1,550 libraries, we found that 8 of the 12 lead ASOs ameliorated the targeted XDP aberrant splicing. Moreover, we found that the two lead ASOs exhibited 38% and 43% rescue of XDP-specific DEGs that were also rescued by CRISPR excision of the SVA (enrichment p-values = 2.06e-13 and 2.27e-05, respectively). These rescues represented restoration of key molecular functions previously implicated in XDP, such as synaptic function, DNA-binding transcription factor activity, and gliogenesis. This study highlights a path to a potential targeted therapeutic for XDP and the capacity to exploit functional genomic signatures in patient-derived neural models to develop a scalable precision therapeutic platform for rare genetic disorders.

Summary Modern molecular biology tools and technologies such as CRISPR have sped up scientific discovery. From an educational perspective, these advancements are both exciting and overwhelming. Educators shaping these future scientists face the ongoing challenge of staying compliant with the latest developments in molecular biology while finding effective ways to teach these discoveries. As the use of CRISPR gene editing technology continues to expand globally, there is an increasing need for a workforce that is both knowledgeable about its theoretical foundations and trained in its practical use. While advanced, technology-driven STEM courses have the potential to improve student retention, they are often lecture-heavy and lack intentional engagement strategies that support deeper learning. Moreover, agriculture is the second most impacted sector by this technology, yet there is a significant lack of teaching materials focused on CRISPR in plant biology. To address these gaps, we developed a framework for teaching gene editing that incorporates multiple engagement strategies beyond traditional lecture-based instruction. This framework was implemented over two semesters in an Introduction: to Gene Editing course at Tennessee State University, offered to both undergraduate and graduate students enrolled in a degree in Agricultural Sciences. This manuscript outlines the various strategies used in the course including active learning, multimodal instructional approaches and experiential learning strategies that can be adopted in other classrooms to effectively teach gene editing. Survey-based results from the course indicate a measurable increase in student comfort with designing and executing CRISPR-Cas based experiments. Societal Impact Statement Plant biology lacks accessible teaching materials for CRISPR, a powerful gene-editing technology widely used to improve agriculture. We developed an engaging framework to teach CRISPR concepts to advanced undergraduates and graduate students in plant sciences, which can be readily adopted by other instructors. The approach increased self-reported confidence in students and comfort in explaining CRISPR. Since instructors often have limited time to design interactive lessons, this framework offers a ready-to-use, effective strategy that makes CRISPR more widely available in classrooms, ultimately strengthening CRISPR literacy in the future agricultural workforce.

ABSTRACT Diatoms are major phytoplanktonic algae with secondary endosymbiotic plastids that differ in cellular and regulatory traits from those of the green lineage. Here we exploited the heterotrophic growth ability of Cyclotella cryptica to create the first diatom photosynthetic mutant by CRISPR-Cas inactivation of the nucleus-encoded ATP synthase subunit γ. These mutants showed impaired phototrophic capacity and altered thylakoids morphology. In absence of γ-ATP, protons that accumulate in the thylakoid lumen slow down the cytochrome b 6 f complex, thus keeping the electron carriers downhill oxidized. These results, reversible when the proton gradient is suppressed, demonstrate the existence of a photosynthetic control in diatoms. At variance with the wild type, γ ATP synthase mutants cannot grow heterotrophically in darkness nor in the light when photosystem II is inhibited. This requirement of heterotrophic growth on photosynthetic electron transfer or on the presence of plastidial ATP synthase suggests that the proton motive force (pmf) is a central integrator of the metabolic interaction between photosynthesis and heterotrophy. Our results establish C. crytica as a robust model for analysis of photosynthetic function, regulation and metabolic integration in organisms with secondary plastids. Teaser Mutagenesis in the facultative autotroph diatom Cyclotella cryptica enables exploration of essential plastid functions in diatoms

ABSTRACT Neuronal loss is a hallmark of neurodegeneration and brain injury. Direct reprogramming of astrocytes into neurons has emerged as a promising approach to restore lost neurons. Comprehensive mapping and characterization of candidate astrocyte-to-neuron reprogramming factors is an essential step to realizing the potential of this strategy. Here, we established a CRISPR activation (CRISPRa)-based approach for neuronal reprogramming of primary human astrocytes. We conducted high-throughput CRISPRa screens of all human genes encoding transcription factors (TFs) to identify novel and efficient reprogramming factors. scRNA-seq characterization of top hits revealed that single TFs reprogram primary human astrocytes into multiple neuronal subtypes with distinct cell type-specific gene signatures. We demonstrate that INSM1 reprograms astrocytes to a glutamatergic neuron-like state and has broad neurogenic activity across different cell types and across human and mouse contexts. Finally, we conduct paired CRISPRa screens to identify cofactors that cooperate with INSM1 to enhance neuronal reprogramming and subtype specification, and elucidate genomic mechanisms of interaction and downstream regulators.

Variants in the LRRK2 and GBA1 genes are among the most common risk factors associated with Parkinson’s disease (PD). Both patients carrying PD-associated variants in GBA1 , encoding lysosomal enzyme glucocerebrosidase (GCase), and a subset of non-carrier patients have been shown to have reduced GCase enzymatic activity, suggesting that reduced GCase activity may be a feature of both genetic and a subset of sporadic PD. However, the effect of PD-associated variants in LRRK2 , encoding a serine/threonine kinase, on GCase activity remains controversial, with conflicting results in various tissues and cell types. Moreover, rare patients carrying both GBA1 and LRRK2 risk alleles seem to have a more benign disease course than carriers of GBA1 variants alone, suggesting a complex interplay between these two genes in PD. Here we evaluate the effect of LRRK2 kinase activity on GCase activity in human induced pluripotent stem cell (iPSC)-derived microglia (iMGs), a PD-relevant brain cell type expressing high levels of LRRK2. Using CRISPR editing, isogenic control iPSC lines were generated to match PD patient-derived iPSC lines harbouring the LRRK2 p.G2019S, p.M1646T, or p.N551K-p.R1398H protective haplotype variants. Whereas iMGs harbouring the p.M1646T variant, and the protective haplotype, respectively increased and decreased phosphorylation of canonical LRRK2 substrate, Rab10, GCase protein levels and activity were not altered in any of the LRRK2 variant lines. Additionally, whereas pharmacological inhibition of LRRK2 kinase activity had no impact on GCase activity in iMGs under basal conditions, it attenuated the increase in GCase activity elicited in response to interferon γ (IFNγ) treatment. Moreover, GCase activity induced by IFNγ was reduced in PD risk LRRK2 p.M1646T iMGs and increased in p.N551K-p.R1398H protective haplotype iMGs compared to their isogenic corrected controls, congruent with their respective effects on LRRK2 kinase activity and PD risk. Thus, our data suggests a role for LRRK2 kinase activity in regulation of GCase activity in response to neuroinflammation.

Despite substantial insight into mechanisms underlying arterial blood vessel development, multiple aspects of vein development remain elusive, including vein-determining extracellular signals and cell-fate trajectories 1–10 . One might expect arteries and veins to arise simultaneously during development, as both are needed for a functional circulatory system. Nevertheless, arteries often precede veins in vivo , as exemplified by the first intraembryonic blood vessels 11–17 . Here we present a model of vein differentiation that answers longstanding questions in the field. By reconstituting human vein endothelial cell (EC) differentiation from mesoderm in vitro , we discovered that vein development unfolds in two steps driven by opposing signals. First, VEGF is necessary to differentiate mesoderm into “pre-vein” ECs—a newly-defined intermediate state—and to endow endothelial identity. Second, once cells have acquired pre-vein EC identity, VEGF/ERK inhibition is necessary to specify vein ECs. Pre-vein ECs co-expressed certain arterial (SOX17) and venous (APLNR) markers and harbored poised chromatin at future venous genes. However, VEGF/ERK inhibition was necessary to activate poised venous genes (e.g., NR2F2 ), and for pre-vein ECs to complete venous differentiation. Intersectional lineage tracing supported a pre-vein intermediate step in vivo : early Sox17+ Aplnr+ ECs also formed veins in mouse embryos. We leveraged this developmental knowledge for disease modeling by differentiating human pluripotent stem cells into artery and vein ECs, and comparing their responses to Ebola and Andes viruses under biosafety-level-4 containment. Artery and vein ECs responded divergently to the same virus, thus revealing that developmentally specified cell identity impacts viral infection. Taken together, we propose a two-step model for vein development wherein VEGF first differentiates mesoderm into pre-vein ECs, but subsequent VEGF/ERK inhibition generates vein ECs. VEGF activation is thought to be broadly essential for vascular development 6,18 , and thus our discovery that VEGF/ERK inhibition specifies vein identity has potential implications for understanding current therapies that either activate or inhibit VEGF signaling 19,20 .

ABSTRACT Extrachromosomal DNA (ecDNA) is a prevalent and devastating form of oncogene amplification in cancer 1,2 . Circular megabase-sized ecDNAs lack centromeres and segregate stochastically during cell division 3–6 yet persist over many generations. EcDNAs were first observed to hitchhike on mitotic chromosomes into daughter cell nuclei over 40 years ago with unknown mechanism 3,7 . Here we identify a family of human genomic elements, termed retention elements, that tether episomes to mitotic chromosomes to increase ecDNA transmission to daughter cells. We develop Retain-seq, a genome-scale assay that reveals thousands of human retention elements conferring generational persistence to heterologous episomes. Retention elements comprise a select set of CpG-rich gene promoters and act additively. Live-cell imaging and chromatin conformation capture show that retention elements physically interact with mitotic chromosomes at regions which are mitotically bookmarked by transcription factors and chromatin proteins, intermolecularly recapitulating promoter-enhancer interactions. Multiple retention elements are co-amplified with oncogenes on individual ecDNAs in human cancers and shape their sizes and structures. CpG-rich retention elements are focally hypomethylated; targeted cytosine methylation abrogates retention activity and leads to ecDNA loss, suggesting that methylation-sensitive interactions modulate episomal DNA retention. These results highlight the DNA elements and regulatory logic of mitotic ecDNA retention. Amplifications of retention elements promote the maintenance of oncogenic ecDNA across generations of cancer cells, revealing the principles of episome immortality intrinsic to the human genome.

The C1858T PTPN22 (R620W) variant has been implicated in the pathogenesis of several autoimmune disorders and represents a promising immunotherapeutic target for Type 1 diabetes. We have been implementing a novel immunotherapeutic approach based on the use of lipoplexes that deliver siRNA duplexes. Efficacy and safety of lipoplexes halting variant expression was demonstrated in the peripheral blood of patients in vitro. According to regulatory authorities in Europe preclinical safety and efficacy must be ascertained in vivo in appropriate animal models before undertaking clinical investigations. In the light of the foregoing the aim of this study was to verify that lipoplexes against the murine Ptpn22-R619W, equivalent to the human PTPN22-R620W, could be used for animal experimentation. The murine fibroblast cell line L929 was transfected with the PF62-pLentiPtpn22-R619W plasmid. We designed siRNA duplexes specific for the Ptpn22-R619W allele and formulated them into cationic lipoplexes in order to halt the variant expression in the transfected L929 cell line. Transfection of fibroblasts expressing R619W using lipoplexes resulted in efficient silencing at 100 pmol siRNA after 48 hours post-transfection reaching higher significant knock‑down after 72 hours. Lipoplexes efficiently suppress the pathogenic Ptpn22 variant expression in vitro, supporting the feasibility of a pre‑clinical platform for in vivo lipoplexes testing in CRISPR‑engineered NOD/ShiLtJ mice carrying the R619W mutation.

Abstract Background Vitamin K 2 (VK 2 ), as a derivative of the menaquinone family, plays an important role in the prevention of osteoporosis and cardiovascular calcification. The realization of the industrialization of VK 2 and the reduction of its production cost have become the focus of attention. Results In this work, an E. coli strain with high VK2 accumulation was constructed through rational metabolic engineering and stepwise improvement based on regulatory metabolic information and CRISPR/Cas9-mediated gene knockout. We first constructed a recombinant E. coli to produce menaquinol-8 (MKH 2 -8, a reduced form of VK 2 ) by overexpressing menA and ubiE , the rate-limiting enzymes of the menaquinol pathway. Secondly, we overexpressed different related genes wrbA , qorB and menF , respectively. Among these recombinant strains, the strain MUW reached a yield of 301.96 mg/L after 48 h of fermentation. The optimization of the medium led to an increase in the accumulation of VK 2 . Subsequently, the rational metabolic engineering of gene knockout further increased the VK 2 yield. The recombinant strain ΔB:MUW was selected as the dominant strain for further optimization, with a high VK 2 yield of 723.59 mg/L. A final attempt is to overexpress ispB gene to increased flux of isoprenoid side chain synthesis. After 48 h cultivation, a high VK 2 yield of 1355.29 mg/L was achieved by ΔB:MUWI in a 5 L fermenter. Conclusions This study demonstrates that metabolic engineering techniques combining rational modification of the metabolic pathway and optimization of gene expression can effectively cultivate strains with industrial competitiveness.

We have recently found that by promoting transcriptional elongation, histone deacetylase inhibitors (HDACi) cooperate with the antisense oligonucleotide nusinersen (ASO1) to upregulate exon 7 (E7) inclusion into SMN2 mRNA (Marasco et al., 2022). In parallel, ASO1 also elicits the deployment of the silencing histone mark H3K9me2 on the SMN2 gene, creating a roadblock to RNAPII elongation that downregulates E7 inclusion. By removing the roadblock, HDACi counteract the undesired chromatin effects of the ASO, resulting in higher E7 inclusion, which allowed us to propose a combined therapy for spinal muscular atrophy (SMA). We show here that the histone methyl transferase G9a is involved in the ASO1-elicited deployment of H3K9me2 not only at the SMN2 E7 region, located towards the 3’ end of the gene, but also at its promoter. Furthermore, using a CRISPR/dCas-based (dead-Cas9-based) strategy, we show that targeting H3K27 histone acetylation at the SMN2 E7 region duplicates the HDACi effect, which overcomes potentially pleiotropic effects. Most noticeably, when acetylation is targeted to the E7 region alone, the 30-kbp-distant promoter becomes acetylated and activated. These cross-talks between the two ends of the SMN2 gene prompted us to use chromosome conformation analysis (3C) to find that ASOs can promote gene looping. This novel property of ASOs depends on cohesin and may explain promoter activation by distant alternative splicing events. Abstract Figure In brief Antisense oligonucleotides (ASOs) modulate alternative splicing through base-pairing to sequences in the pre-mRNA. Simultaneously, they may promote the deployment H3K9me2 marks (purple diamons) and looping between the promoter and the 3’end region of a gene, with subsequent transcriptional activation.

The Icelandic mutation in the amyloid precursor protein (APP), APP A673T , has been identified in Icelandic and Scandinavian populations and is associated with a significantly lower risk of developing Alzheimer’s disease (AD). Although this mutation led to reduction in amyloid β-protein (Aβ) production, its effect on tau pathology is not well studied. We have crossed line 66 (L66) tau transgenic mice that overexpress the P301S aggregation-prone form of tau with C57Bl6/J mice expressing a single point mutation edited into the murine APP gene via CRISPR-Cas gene editing, termed APP A673T . We have performed ELISA, histopathological and behavioural analyses of heterozygous male/female L66 and L66xAPP A673T crosses at the age of 6 months to investigate the effect of the A673T mutation on tau brain pathology and behavioural deficits in these mice. Using immunohistochemistry, we found only a moderate, yet significant, reduction of mAb 7/51-reactive tau in prefrontal cortex for L66xAPP A673T compared to L66 mice. Quantification of tau in soluble/insoluble brain homogenate fractions by ELISA confirmed the lack of overt differences between genotypes, as did our extensive behavioural phenotyping using six different paradigms accessing motor function, olfaction, depression/apathy-like behaviour, as well as exploration and sociability. Therefore, the APP A673T mutation does not appear to modulate tau pathology or motor and neuropsychiatric behaviour in L66 tau transgenic mice.

ABSTRACT Accurate genome assemblies are critical for understanding small RNA-mediated genome defense. In animals, the PIWI-interacting RNA (piRNA) pathway protects genome integrity by silencing transposable elements. Studying how piRNAs are generated and how they guide heterochromatin formation requires complete reconstruction of genomic piRNA source loci and detailed transposon maps. Here, we present a high-quality de novo genome assembly of Drosophila melanogaster ovarian somatic cells (OSCs), a widely used cell line that recapitulates nuclear piRNA biology. The OSC genome differs substantially from the reference genome, with major differences in transposon content and piRNA cluster composition. Our assembly resolves the 700 kb flamenco locus, the primary piRNA cluster in OSCs, and provides a genome-wide transposon map. Using this resource, we characterize piRNA source loci, reveal how piRNA cluster composition determines transposon-derived piRNA profiles, and clarify the widespread impact of the nuclear piRNA pathway on heterochromatin. Finally, we provide an open platform for integrating user-generated datasets with the OSC genome, creating a community resource for studying transposon control and piRNA biology.

ABSTRACT T-cell based immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy face substantial hurdles when confronting solid tumors such as ovarian cancer, where metabolic constraints in the tumor microenvironment limit T cell infiltration and function. In particular, T cells exposed to nutrient deprivation and hypoxia upregulate autophagy, a lysosomal degradation pathway that negatively regulates effector responses. Here, we used CRISPR-Cas9 to target a folate receptor alpha (αFR) CAR expression cassette into the locus of the essential autophagy gene ATG5 , thereby generating autophagy-deficient CAR-T cells in a single editing step. Targeted metabolite profiling revealed that deletion of ATG5 induced widespread metabolic reprogramming characterized by increased glucose and amino acid uptake. Functionally, ATG5 -knockout CAR-T cells maintained high cytolytic activity when assayed in patient-derived ascites in vitro , and exhibited superior and long-lasting tumor control against ovarian tumors in vivo . Taken together, our results suggest that deletion of ATG5 metabolically primes CAR-T cells for enhanced cytotoxicity in immune-suppressive conditions, thereby improving the therapeutic potential of αFR CAR-T cells for ovarian cancer immunotherapy.

Abstract Purpose Osteosarcoma (OS) is a prevalent primary malignant bone tumor that predominantly affects children, adolescents, and young adults. Proline-rich protein 11 (PRR11) is wellknown for its role in regulating cell cycle progression and promoting tumorigenesis. Nevertheless, the precise molecular mechanisms underlying PRR11-driven tumorigenesis in OS have yet to be elucidated. In the present study, we aimed to elucidate the role of PRR11 in OS and its underlying molecular mechanisms. Methods Genotype‒tissueexpression(GTEx) and The Cancer Genome Atlas (TCGA) data were analyzed for PRR11 expression (normal vs OS) and survival differences (low vs high expression). Immunohistochemistry(IHC) and western blotting(WB) were performed to examine the expression distribution of the PRR11 protein in OS tissues and cell lines. Three types of lentiviral vectors were used to establish stable 143B cell lines: (1) miRNA-based shRNA vectors, (2) Lenti-CRISPR-Cas9 vectors, and (3) overexpression vectors. RNA-seq analysis of the miRNA-based shRNAs. WB was used to elucidate the mechanisms by whichPRR11 affects DNA damage, DNA repair, the cell cycle, and the Hippo signaling pathway. Moreover, functional assays included colony formation, wound healing, and transwell assays in vitro and subcutaneous inoculation in vivo . Results This studyidentified PRR11 as a pivotal regulator that promotes OS cell migration, invasion, and proliferation in vitro and promotes OS subcutaneous inoculation. RNA-seq analysis revealed that PRR11 silencing regulates several signaling pathways, including the cell cycle, the DNA damage response, and DNA repair;subsequently, the detection of DNA damage/repair markers and cell cycle-related proteins further confirmed alterations in these signaling pathways. Subsequent flow cytometry experiments revealed that PRR11 could prolong the G0/G1 phase and shorten the G2/M phase. Conclusions PRR11 functions as an oncogene in OS, where the PRR11-Hippo axis drivestumor progression through a DNA damage-cell cycle coupling mechanism.

CRISPR-Cas9 is a gene editing tool used extensively in biological research that is now making3 its way into clinical therapies. With the first CRISPR therapy obtaining approval by the United4 States’ Food and Drug Administration (FDA) in late 2023, we look at clinical trials of emerging5 therapies involving CRISPR-Cas9, currently the most prevalent CRISPR-based tool in these6 trials. A CRISPR-based therapy is currently approved for treatment of both sickle-cell anemia and7 transfusion-dependent β-thalassemia but clinical trials for CRISPR-based therapeutics include a8 much broader range of targets. CRISPR-Cas9 is being explored to treat cancer, infectious disease,9 and more. This review highlights CRISPR-Cas9 clinical trials registered at clinicaltrials.gov as of10 12/31/2024.

Background In this study, we aim to develop a yearly updatable database that could predict chemotherapeutic drug resistance and overall survival probability in breast cancer patients. Existing drug sensitivity databases depend on correlation-based predictions. In our study, candidates involved in drug resistance are chosen based on cell line validation (overexpression or downregulation or inhibition of candidates) studies, curated manually. Method 28,773 mRNA expression signatures from 914 breast cancer patients were extracted from cProsite. 106 of these patients had clinical information and log2 fold change information required for this study. We categorized these patients into deceased and surviving groups from TCGA. To prepare a database that can predict drug resistance and overall survival, we included mRNAs that were over-expressed in at least 80% of the breast cancer patients and mRNAs over-expressed in deceased and surviving groups. In addition, we also reported breast cancer-associated drug resistance candidates which have been reported in cell-line based studies. The database matrix preparation involved an approximate of 15000 manual searches of cell validated studies. (750 candidates x 20 drugs). The database was validated using a publicly available breast cancer patient proteomics data. Results Our analysis identified a list of top priority candidates associated with multidrug resistance, categorized based on their resistance to >15 drugs, 5-15 drugs, and 2-4 drugs. Analysis of patient profiles in the database revealed that the number of proteins contributing to drug resistance was high in the poor prognosis category compared to the good prognosis category. Conclusions Our study highlights the probable gaps in breast cancer drug resistance research, as only a small subset of overexpressed mRNA candidates found in patients are studied in vitro or in vivo experiments focusing on drug resistance. We also identified candidates involved in multidrug resistance, whose role in drug resistance has not been studied in more than 15 drugs. After further validations, this will benefit the clinicians and upcoming CRISPR gene therapeutics.

Parkinson’s disease (PD) is a devastating neurodegenerative disorder primarily characterized by the progressive and unstoppable loss of dopaminergic (DA) neurons in the substantia nigra. We previously identified NATO3 (FERD3L), a conserved developmental transcription factor, as essential for maintaining DA neuron function during aging. Here, we show that AAV-mediated Nato3 gene transfer into the mouse substantia nigra prevents DA neuron degeneration in both MPTP-induced and α-synuclein (α-Syn) overexpression PD models. This neuroprotective effect is achieved by improving autophagic flux and ⍺ -Syn clearance. Furthermore, lentiviral-mediated NATO3 overexpression in human midbrain DA neurons, derived from induced pluripotent stem cells carrying the pathological ⍺ -Syn A53T mutation, effectively reversed key disease hallmarks. These include ⍺ -Syn accumulation, aberrant mitochondrial morphology, autophagic impairments, and compromised neurite structure. Collectively, these in vivo and in vitro findings highlight NATO3’s role in safeguarding DA neurons against pathological cellular events, positioning NATO3 as a therapeutic target for PD.

ABSTRACT Heterogeneous T cell states are critical in immune responses and have been explored by CRISPR-based and synthetic domain-swapped transcription factor (TF) screens, yielding novel insights and immunotherapeutics. However, a scalable strategy to map TFs in primary human T cells is lacking, which limits our understanding of the functions of critical TFs. We therefore adapted a transposon-based TF mapping strategy termed Calling Cards for primary human CD8 T cells, applying it to five key TFs with undefined binding sites in this cell type: TOX, TOX2, TCF7, SOX4, and RBPJ. To derive biological insights from these data, we developed an analytical framework to integrate TF binding with multi-omic sequencing data, revealing convergence of TOX and TCF7 binding at dynamic enhancers of memory CD8 T cells. We then identified TF co-bound gene programs related to memory and exhaustion states in addition to putative gene targets of known and unappreciated TF roles, including TOX binding at critical genes of both exhaustion and terminal effector memory differentiation. To further scale our TF analysis platform, we modified Calling Cards to create TFlex a method uniquely suited for multiplexed mapping of paralogous TFs. We applied TFlex to simultaneously map eight natural and domain-swapped TFs in primary human CD8 T cells, which demonstrated that domain-swapped TFs display emergent behavior in binding site selection and transcriptional effects on target genes that cannot be estimated as the sum of their constituent domains. Collectively, our data highlight the importance of scalable TF mapping in primary human T cells to elucidate TF function and the transcriptional regulation of cell states.

Abstract Background The genus Leuconostoc has significant importance in food fermentations and is increasingly evaluated for probiotic applications. Although Leuconostoc falkenbergense is a recently identified species, and its in-depth genomic characterization and probiotic attributes remain largely unexplored, particularly for strains derived from diverse natural sources such as traditional dairy food matrices. This study therefore aimed to isolate and characterize a L. falkenbergense strain (BSMRAU-M1L5) from naturally fermented traditional buffalo milk curd and performed whole-genome sequencing with detailed genomic analyses to evaluate its probiotic potential and safety profile for future applications. Results The draft genome of L. falkenbergense BSMRAU-M1L5 spans 1.78 Mb, assembled into 96 contigs, and encodes 1,844 genes. Phylogenetic and average nucleotide analyses with 21 other L. falkenbergense strains confirmed that BSMRAU-M1L5 is most closely related to strain C, previously isolated from yogurt in China. Pan-genome analysis revealed a substantial core gene set and 124 strain-specific genes in BSMRAU-M1L5, including 99 hypothetical genes, while the remainder were linked to metabolic versatility, survivability, and functional adaptation. Functional annotation revealed genes involved in carbohydrate utilization, vitamin and acetate biosynthesis, acid tolerance, antioxidant defense, stress response, adhesion, AI-2 signaling, exopolysaccharide production, and antimicrobial activity, highlighting its strong probiotic potential. The genome also harbors CRISPR-Cas systems, insertion sequence elements, and prophages, while lacking virulence and biogenic amine–related genes, confirming its genomic safety. Despite harboring three vancomycin resistance genes, the strain was susceptible to most tested antibiotics and exhibited γ-hemolysis, supporting its non-pathogenic nature and overall safety profile. Conclusion The genome of L. falkenbergense BSMRAU‑M1L5 encodes diverse metabolic and probiotic properties, supported by both genomic and phenotypic evidence of safety, positioning it as a promising candidate for use in fermented food production and broader biotechnological applications.

RNA localization and local translation mediate spatial and temporal control of polarized cells, including radial glial cells (RGCs) which produce and organize neurons and glia. Within RGCs, RNAs are transported long distances to basal endfeet, where they can undergo local translation. However, the subcellular composition of RGCs and function of local gene regulation remains largely unknown. Here, we discover that basal endfeet harbor a rich transcriptome including a Dynein component critical for subcellular RGC function. By purifying RGC compartments in vivo , we discover ∼3000 endfoot transcripts, including ∼800 highly enriched compared to cell bodies. Many endfoot-enriched transcripts exhibit conserved subcellular localization in neurons and glia and are associated with neurodevelopmental disease. We show that endfoot-enriched Dync1li2 regulates RGC basal morphology and subsequently interneuron organization. Finally, we develop LOCAL-KD, a CRISPR-Cas13 based method for subcellular mRNA knockdown in vivo . Leveraging this, we demonstrate that endfoot-localized Dync1li2 is critical for RGC morphology. Our study establishes experimental paradigms to understand RNA localization in the nervous system. Moreover, we discover RGCs have a vast subcellular transcriptome, revealing foundational insights into how RGCs control cortical development.

The emergence of multidrug-resistant (MDR) "superbugs" is a serious threat to world health because of the interdependence of environmental ecosystems, agriculture, and human medicine within the single health framework. Horizontal gene transfer (HGT), residue persistence, and excessive antibiotic use have accelerated the development of antimicrobial resistance (AMR) by 2050, it is predicted that AMR would be responsible for 8.22 million deaths. To evade conventional therapies, bacteria employ sophisticated resistance mechanisms such enzymatic inactivation (e.g., β-lactamases, carbapenemases), target alteration (e.g., PBP2a in MRSA), efflux pumps, biofilm formation, and reduced membrane permeability. In light of this dilemma, new strategies are needed to restore the efficacy of antibiotics and stop the spread of resistance. Advances in antibiotic adjuvants, like efflux pump blockers and β-lactamase inhibitors like avibactam, complement existing drugs to combat resistance. The arsenal against MDR pathogens is further diversified by phage therapy, CRISPR-Cas systems, anti-virulence inhibitors, combinatorial therapy and vaccinations. However, challenges persist, biofilm resilience, plasmid-borne anti-CRISPR defences, and ecological risks of gene-editing tools necessitate rigorous mitigation AI-driven diagnostics, metagenomics, and genomics together offer a revolutionary approach to AMR management and surveillance. To address the superbug epidemic & pandemic, the paper integrates the genetic, molecular, and ecological facets of AMR and highlights the vital need for international collaboration, sustainable practices, and One Health-aligned policies.

Intracellular photosymbiosis has evolved across life and forms the foundation of coral reef ecosystems. Using the sea anemone Aiptasia as a model, we generated a high-quality proteome of the symbiosome, the organelle that houses algal symbionts. This proteome revealed protein trafficking mechanisms and the types of biomolecules exchanged during symbiosis. Symbiosomal enrichment of lysosomal proteins, visualization of lysosomal fusion, along with reduced symbiosis following knockdown of lysosomal genes, supports its phagolysosomal identity and that extensive co-option of lysosomal proteins shapes the symbiosome. CRISPR/Cas9-induced mutations in the symbiosomal and lysosomal bicarbonate/sulfate transporter, SLC26A11, disrupted symbiosis in both Aiptasia and a reef-building coral. These findings support that anemones and corals independently evolved a carbon-concentrating and sulfate transport mechanism to fuel photosymbiosis by co-opting an orthologous lysosomal transporter.

ABSTRACT Cancer therapies are typically effective in subsets of patients, reflecting the molecular diversity of cancers and motivating the need for predictive biomarkers of response. Biomarker-guided therapy is increasingly useful in oncology, yet biomarker discovery remains complicated by the large number of molecular features that make it difficult to distinguish causal determinants from spurious associations. To address this challenge, we combined functional genomic screening, proteomics, and drug sensitivity profiling to discover response biomarkers for a number of therapies used in the treatment of Peripheral T-Cell Lymphomas (PTCL). First, we used genome-wide CRISPR-dCas9 interference screens in PTCL cells under drug treatment to identify a shortlist of genes whose knockdown directly increases or decreases drug sensitivity. Next, we profiled drug responses across a diverse panel of 30 PTCL cultures and, from the shortlist, identified genes whose protein abundance correlated with drug sensitivity. Genes detected by both approaches are causal determinants of drug response and correlates of drug response across the panel of lymphoma cultures, making them promising candidates for predictive biomarkers. Basal expression of the reduced folate carrier SLC19A1 was a strong predictor of pralatrexate sensitivity, consistent with its role as the primary transporter for pralatrexate uptake. Simulated clinical trials predicted that biomarker-guided patient selection could improve the power to detect significant benefit of adding pralatrexate to frontline chemotherapy in PTCL. These findings illustrate how the causal insights of functional genetic screens can augment correlative studies to identify biomarkers of drug response, and suggest the potential for precise use of pralatrexate for PTCL.