Stathmin-2 (STMN2) is a microtubule associated protein that plays a role in the stability of microtubules of axons in the nervous system of animals. In this study we generated a novel zebrafish STMN2 knockout (KO) model. STMN2 is represented by two genes in the zebrafish genome: stmn2a and stmn2b . Using the CRISPR/Cas9 mutagenic system we selected founder fish lines harbouring frameshift mutations in both genes and bred these together to generate a double stmn2a and stmn2b KO model. Using these models, we observed increased developmental lethality in our double stmn2a and stmn2b KO model and impaired motor function at larval stages of development. Examination of the larval neuromuscular junction (NMJ) revealed a slight increase in the number of orphaned NMJs in trunk musculature as well as a reduction in amplitude of miniature endplate currents in our double stmn2a and stmn2b KO model. In a final series of experiments, we show impaired ventral root axon regrowth following transection in double stmn2a and stmn2b KO larvae. Our findings suggest that while not essential for motor axon development, loss of stmn2a and stmn2b expression perturbs the assembly of zebrafish NMJs during development resulting in a minor motor phenotype and impairs that ability to regenerate motor axons following injury.

The widespread use of antibiotics promotes both resistance and tolerance. While resistance enables bacterial growth in the presence of drugs, tolerance allows survival during treatment, generating persisters that seed relapse and promote resistance. Despite its clinical relevance, the molecular basis of tolerance remains poorly understood. Using proteomic and metabolomic profiling combined with machine learning, we identified thiol oxidation as a robust predictor of tolerance in the human pathogen Pseudomonas aeruginosa . Single-cell analyses established a direct link between thiol oxidation and drug survival, indicating that redox imbalance drives persistence. Whereas depletion of coenzyme A (CoA), a central thiol-containing metabolite, scaled with tolerance, restoring CoA using engineered catalysts from Staphylococcus aureus abolished tolerance, establishing a causal relation between CoA availability and drug susceptibility. Thiol-based predictors also accurately capture tolerance of clinical P. aeruginosa isolates. These findings establish CoA-centered redox control as a key determinant of tolerance, opening opportunities for diagnostics and therapeutic interventions to prevent infection relapses.

Invasion plasticity allows malignant cells to toggle between collective, mesenchymal and amoeboid phenotypes while traversing extracellular matrix (ECM) barriers. Current dogma holds that collective and mesenchymal invasion programs trigger the mobilization of proteinases that digest structural barriers dominated by type I collagen, while amoeboid activity allows cancer cells to marshal mechanical forces to traverse tissues independently of ECM proteolysis. Here, we use cancer spheroid-3-dimensional matrix models, single-cell RNA sequencing, and human tissue explants to identify the mechanisms controlling mesenchymal versus amoeboid invasion. Unexpectedly, collective/mesenchymal- and amoeboid-type invasion programs – though distinct – are each characterized by active tunneling through ECM barriers, with expression of matrix-degradative metalloproteinases. CRISPR/Cas9-mediated targeting of a single membrane-anchored collagenase, MMP14/MT1-MMP, ablates tissue-invasive activity while co-regulating cancer cell transcriptional programs. Though changes in matrix architecture, nuclear rigidity, and metabolic stress as well as the presence of cancer-associated fibroblasts are proposed to support amoeboid activity, none of these changes restore invasive activity of MMP14-targeted cancer cells. While a requirement for MMP14 is bypassed in low-density collagen hydrogels, invasion by the proteinase-deleted cells is associated with nuclear envelope and DNA damage, highlighting a proteolytic requirement for maintaining nuclear integrity. Nevertheless, when cancer cells confront explants of live human breast tissue, MMP14 is again required to support invasive activity. Corroborating these results, spatial transcriptomic and immunohistological analyses of invasive human breast cancers identified clear expression of MMP14 in invasive cells that were further associated with degraded collagen, underlining the pathophysiologic importance of this proteinase in directing invasive activity in vivo .

Lipid abnormalities are emerging as key pathogenic mechanisms in neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Lewy body dementia. Astrocytes in the brain provide APOE proteins and influence neuronal metabolism and health. Using live cell imaging and objective neurite imaging techniques, we show that following induction of cellular lipid (cholesterol and triglycerides) load by inhibiting the lysosomal cholesterol transport protein NPC1 in human neuron-astrocyte co-cultures, that human astrocytes CRISPR edited to be either APOE3 or 4 variants have different effects on rescuing dystrophic neurites, where axons and dendrites of nerve cells become disfigured. APOE3, but not APOE4 or APOEKO, astrocytes prevented cholesterol and lipid induced neurite damage in APOE4 neurons. In the media of APOE3 co-cultured astrocytes with neurons the HDL-like particles were larger and presumably more lipidated than equivalent APOE4 co-cultures. This discovery highlights that living APOE3 astrocytes control key biological mechanisms by physiologically enhancing lipid cellular homeostasis, that can rescue lipid-induced neurite structural abnormalities relevant to Alzheimer’s disease and neurodegenerative diseases. Significance statement Neurodegenerative diseases like Alzheimer’s (AD) are often defined by abnormal protein aggregates, but growing evidence points to lipid dysfunction as a key driver, especially in APOE4 carriers, the strongest genetic risk factor for AD. We developed a live cell imaging based human cell culture model using isogenic iPSC-derived neurons and astrocytes (APOE3, APOE4, or APOE knockout) to study this. By blocking cholesterol export via NPC1 inhibition, we mimicked lysosomal lipid stress and found that APOE3 astrocytes uniquely protected APOE4 neurons from forming abnormal neurite swellings. These APOE3 astrocytes produced larger HDL-like particles than APOE4 that supported neuronal lipid balance. Our results show that APOE3 astrocytes can rescue APOE4-related cellular dysfunction, offering a potential path for therapy and biomarker discovery.

ABSTRACT Female infertility represents a significant challenge in reproductive medicine. Although it is known to be primarily due to oogenic and early embryonic failures, the molecular mechanisms underlying these failures remain elusive. The Piwi-piRNA pathway is crucial for gametogenesis in diverse organisms. Yet, its role in mammalian female fertility is unclear. This is partly because most studies are done in mice, which contain only PIWIL1, PIWIL2, and PIWIL4 that are essential for spermatogenesis but not for oogenesis. PIWIL3 emerges in higher mammals and is highly expressed in human oocytes. Interestingly, female PIWIL3 -knockout golden hamsters exhibit only reduced fertility without detectable defects in oogenesis. This has left the function of PIWIL3 in higher mammals, including humans, unexplored. Here, we discovered that PIWIL3 in the rabbit ( Oryctolagus cuniculus ) shares high homology with human PIWIL3. It is the predominant PIWI protein in oocytes in both species. Using CRISPR–cas9-mediated knockout, we demonstrated that rabbit PIWIL3 is essential for female fertility. Its loss leads to severe defects in oogenesis. Moreover, embryos lacking maternal PIWIL3 arrest developmentally at the 8-cell stage. Mechanistically, rabbit PIWIL3 binds ∼18-nucleotide piRNAs, mirroring the behavior of human PIWIL3, and is critical for piRNA biogenesis. Moreover, it regulates transcriptomic and proteomic landscapes and silences a broad array of transposons during late oogenesis yet activates another set of transposons during early embryogenesis. These findings establish PIWIL3 as a pivotal dual regulator of gene expression and transposon activity, which is essential for oogenesis and early embryogenesis in non-rodent mammals, potentially including humans.

Osteocytes play critical roles in bone, making them attractive targets for therapeutics to improve bone mass and strength. The genes driving osteocyte maturation and function are not fully understood. Here we aimed to identify novel genes responsible for osteocyte differentiation and dendrite development by performing a genome-wide CRISPR-interference (CRISPRi) screen in the Ocy454 osteocyte-like cell line. We identify CD61 (integrin β3) as a marker of osteocyte maturation: surface CD61 expression increases during osteocyte maturation, and CD61 high cells express higher levels of osteocyte marker genes. We then developed a flow cytometry-based assay to quantify surface CD61 protein levels as a phenotypic endpoint for functional genomic screening. In a genome-wide screen, we identified Clip2, which encodes a microtubule binding protein, as one of dozens of genes necessary for CD61 expression. Clip2 inhibition decreased surface CD61 expression, reduced expression of osteocyte-specific genes Dmp1 and Sost , and impaired dendrite morphology in vitro . Together, these results highlight the utility of surface CD61 as a marker of osteocyte maturity and identify a role of the microtubule cytoskeleton for osteocyte differentiation, form, and function.

iNKT cells are emerging as a highly promising immunotherapy platform for the treatment of cancer. To maximise the anti-cancer activity of CAR-iNKT against the blood cancer multiple myeloma we investigated optimal CAR designs and their combination with novel iNKT-specific engagers. We find that amongst five different CAR endodomains, underpinned by increased avidity and a cross talk between Plexin D1 on CAR-iNKT and Semaphorin 4A on myeloma cells, BCMA CD28z CAR-iNKT exert the highest anti-myeloma activity. Notably, CD28z CAR-iNKT outperform their CAR-T counterparts. To expand the anti-myeloma potential of CAR-iNKT, we designed and validated a high efficacy BCMA iNKT-specific engager which exerts significant anti-myeloma activity in conjunction with adoptively transferred iNKT cells. Finally, combined, dual target therapy with FCRL5 CAR-iNKT and BCMA iNKT engagers outperforms FCRL5 CAR-iNKT and limits immune escape of FCRL5-negative myeloma. Thus, optimised iNKT-based, dual-target, dual-modality immunotherapy has enhanced anti-tumor activity against multiple myeloma and potentially other malignancies. Abstract Figure

Background Investigating the subcellular distribution of proteins is crucial for understanding complex cell behaviours and disease mechanisms. Fluorescence microscopy is a key tool for visualising protein localisation. However, its application is often hindered by the lack of high-quality antibodies, and the common approach to overexpress recombinant fusion constructs tagged with fluorescent proteins can introduce artefacts. Endogenous protein tagging, where the sequence for a tag (typically a peptide or fluorescent protein) is integrated into the native genetic sequence encoding a protein of interest, can be used to overcome these limitations, enabling proteins to be visualised without the need for antibodies against the target protein and avoiding complications associated with recombinant protein overexpression. ORANGE (Open Resource for the Application of Neuronal Genome Editing) is a CRISPR-Cas9-based endogenous protein tagging technique which relies on homology-independent targeted integration (HITI)-mediated gene editing. Utilising HITI as the DNA repair pathway of choice gives ORANGE the advantage of being less error-prone than classical homology-directed repair (HDR)-based endogenous protein tagging techniques and additionally, means it can be used in post-mitotic cells. Results We applied the ORANGE system to tag three proteins, CYFIP1, JAKMIP1, and STAT3, and confirmed that the expressed fusion proteins demonstrate expected subcellular localisations through fluorescence microscopy. Unexpectedly, the efficiency of ORANGE editing was less than 1% in HEK293 cells, despite high transfection efficiency. To improve the editing efficiency associated with ORANGE, we combined the ORANGE method with an established Sleeping Beauty transposase/CRISPR-Cas9 fusion technique, which has been shown to enhance HITI-mediated gene editing. Using this new method, which we term Sleeping ORANGE, we successfully tagged CYFIP1 with the fluorescent protein mNeonGreen. Importantly, through fluorescence microscopy and flow cytometry, we demonstrate that Sleeping ORANGE increased gene-editing efficiency by approximately 4- to 6-fold compared to the original ORANGE technique. Conclusions We have developed a method to improve the editing efficiency associated with the ORANGE technique, and with further research to ensure that the fluorescent tags are correctly inserted into their target gene without off-target effects, the Sleeping ORANGE technique may form a valuable tool for researchers to use to better study protein subcellular localisation and dynamics.

Abstract Chimeric antigen receptor (CAR) T cells have transformed cancer therapy, yet many tumors remain refractory. To uncover broadly acting mechanisms of resistance, we performed genome-wide CRISPR activation screens across diverse cancer cell types. These screens converged on RNF19B, an E3 ubiquitin ligase whose high expression correlates with poor patient survival and confers robust CAR-T resistance in mouse xenograft models. Mechanistically, RNF19B destabilizes the interferon-γ receptor subunit IFNGR1, blunting interferon-γ signaling, and simultaneously induces CAMKK2, which mediates resistance through an independent pathway. Pharmacologic inhibition of CAMKK2 synergized with CAR-T therapy in different xenograft mouse models. Our findings identify RNF19B as a previously unrecognized, dual-pathway mediator of CAR-T resistance and reveal CAMKK2 inhibition as a potential strategy to enhance CAR-T efficacy.

Backgrounds The cellular slime mold Dictyostelium discoideum is a widely used model system for studying basic processes in cell and developmental biology. While genetic tools, such as targeted gene disruption by homologous recombination and genome editing using CRISPR/Cas9, are well-established in D. discoideum , efficient methods for conditional loss-of-function studies are limited. Here, we developed a nanobody-based degron system for D. discoideum based on ALFA-tagged protein recruitment to the Skp1-Cullin-F-box (SCF) complex. Results ALFA-tagged Histone H1 was efficiently degraded by expressing anti-ALFA nanobody (NbALFA) fused to the D. discoideum FbxD F-box domain (‘dictyGrad-ALFA’). Cell type-specific targeting was achieved by expressing dictyGrad-ALFA under prestalk- and prespore-specific gene promoters. Furthermore, targeting of adenylyl cyclase A (ACA) resulted in the expected aggregation-deficient phenotype, validating the efficacy of dictyGrad-ALFA-mediated protein depletion. Cell type-specific ACA degradation delayed development but eventually resulted in normal fruiting bodies. Our ALFA-tag approach was further used for conditional knockdown in combination with the auxin-inducible degron 2 (AID2) system, which relies on indole-3-acetic acid (IAA)-dependent binding between NbALFA-mAID and a OsTIR-F-box-Skp1A fusion protein. We obtained efficient IAA-induced degradation in prestalk cells; however, efficiency was low in other cell types. Conclusions Together, these systems pave the way for conditional and cell type-specific protein degradation in D. discoideum , enabling functional analyses of essential genes for development and survival.

SUMMARY The transition zone (TZ) is a selective barrier that maintains ciliary compartmentalization by controlling protein entry and exit. Cilia assembly requires the crossing of this barrier by intraflagellar transport (IFT) trains, scaffolded by IFT-A and IFT-B complexes, which move cargo bidirectionally using kinesin-2 and dynein-2 motors. In Caenorhabditis elegans , IFT-A loss abolishes retrograde transport, resulting in truncated cilia packed with IFT material. Here, we show that blocking TZ assembly prevents dynein-2 and IFT-B accumulation inside IFT-A-deficient cilia and partially rescues axoneme length. Single-particle imaging reveals that this rescue occurs without recovery of retrograde IFT. Instead, IFT particles exit cilia by passively diffusing through the disrupted TZ. Moreover, IFT-A/TZ double mutants shed ciliary extracellular vesicles (EVs) abnormally enriched in IFT components, providing a second clearance route. We conclude that TZ removal alters ciliary responses to retrograde transport defects, promoting diffusion and EV release to clear IFT machinery and facilitate axoneme extension. Highlights - TZ loss provides alternative routes for clearing IFT machinery stalled in IFT-A mutant cilia - Axoneme extension is possible without retrograde IFT when the TZ barrier is removed - Disrupted TZ enables exit of IFT particles by passive diffusion in retrograde IFT-deficient cilia - Excess IFT machinery is discarded in ciliary EVs when retrograde IFT and gating are compromised

ABSTRACT Air sampling is a non-invasive alternative to individual testing for respiratory pathogens. Alternative methods to the “gold standard” quantitative RT-PCR (qRT-PCR) are required to enable higher throughput, lower cost, and more multiplexed detection of pathogens. The multiplexed CRISPR-Cas13 CARMEN Respiratory Viral Panel (RVP) was described previously for high-throughput detection of nine respiratory pathogens from nasal swab samples. Here, we modified and optimized the CARMEN RVP assay to overcome the unique challenges of air samples, including low biomass and environmental inhibitors. We monitored for SARS-CoV-2 and influenza A (Flu A) via qRT-PCR in air samples from 15 schools within Dane County, Wisconsin (USA) during the 2023-2024 school year. SARS-CoV-2 was detectable throughout the entire sampling period, while Flu A detection was seasonal from November 2023 to March 2024. We then analyzed a subset of samples from seven schools using an optimized CARMEN RVP assay for air surveillance (RVP_air) and compared results to qRT-PCR. The RVP_air assay detected several additional pathogens beyond our primary targets. The frequencies and patterns of SARS-CoV-2 positivity, but not Flu A, were similar between qRT-PCR and RVP_air across the 2023-2024 sampling period. We developed a secondary panel (RVP_air_flu) to better detect both H1N1 and H3N2 subtypes. Finally, we compared air sample results to clinical nasal swabs collected from the same school district. For several pathogens (SARS-CoV-2, HCoV-OC43, Flu A), positive air detections coincided with positive nasal swabs. These findings demonstrate that the RVP_air assay can effectively detect airborne pathogens from infected individuals within indoor spaces. IMPORTANCE Air sampling offers a cost-effective alternative to individual testing for respiratory pathogens within congregate settings. Optimization and use of multi-pathogen assays are especially valuable for capturing the breadth of pathogens that may be present simultaneously in the same space. The modified CARMEN RVP assays (RVP_air and RVP_air_flu) detected SARS-CoV-2 and Flu A during similar sampling time periods compared to qRT-PCR, while also detecting several additional respiratory pathogens (seasonal Coronaviruses, Respiratory Syncytial Virus). Importantly, pathogens detected from air samples corresponded to those detected from nasal swabs collected from individuals in the same spaces. Together, these findings highlight the utility of the RVP_air and RVP_air_flu assays as alternatives to qRT-PCR for environmental surveillance, with applications extending to other congregate spaces (hospitals, long-term care facilities) and high-risk settings, better informing communities and improving public health.

ABSTRACT Protein S- acylation is a lipid-based, often reversible post-translational modification that can regulate many aspects of protein behavior, including subcellular localization, protein-interactions, and activity. Emerging evidence has identified roles for individual protein acyltransferases encoded by the ZDHHC in cancers, yet the roles of de- S- acylation enzymes are less clear. Recent evidence suggests that acyl-protein thioesterase (APT1)/ LYPLA1 can impact epithelial-mesenchymal transition and metastasis. This study integrates patient datasets, CRISPR dependency data, and in vitro assays to find APT1 as a context-dependent vulnerability in triple-negative breast cancer (TNBC). Despite the highest protein abundance in luminal MCF7 cells, basal-like MDA-MB-468 cells exhibited the most prominent specific APT1 activity, reflecting subtype-specific regulation. Inhibition of APT1 with ML348 increased S -acylation of nuclear and mitochondrial proteins without altering global acylation. Functionally, APT1 inhibition reduced cell proliferation while inducing minimal apoptosis, consistent with cytostatic growth arrest. Cell-cycle analysis revealed G1 accumulation and reduced S/G2 transition, linking proteomic changes to impaired replication. These findings establish APT1 as a regulator of TNBC proliferation through dynamic de- S- acylation of cell-cycle and mitochondrial proteins, highlighting it as a potential therapeutic vulnerability in aggressive breast cancers.

BRCA1 -deficient ovarian cancer cells undergo extensive metabolic reprogramming, yet the network-level dynamics underlying their proliferation and treatment response remain poorly resolved. Here, we construct large-scale multi-omics-driven kinetic model populations of ovarian cancer metabolism to track how tumor cells adapt to changes in nutrient use, energy production, and metabolite dynamics over time. Across BRCA1 wild-type and mutant cells, these models expose distinct metabolic strategies shaped by transcriptional regulation and prioritize 28 enzyme-mediated vulnerabilities, including 24 linked to existing experimental or approved drugs and 4 previously uncharacterized targets in nucleotide and lipid synthesis. They further recapitulate a ceramide-linked metabolic stress signature shared across diverse chemotherapies. Mechanistic analysis traces the effects of BRCA1 loss to transcription-factor-mediated shifts in enzyme activity, outlining regulatory routes for network-level rewiring. Beyond ovarian cancer, this framework offers a generalizable blueprint for predicting metabolic vulnerabilities, drug responses, and adaptive mechanisms across diverse cancer and metabolic disease contexts. By coupling dynamic metabolism to therapeutic prediction, it delivers actionable hypotheses for biomarker discovery, patient stratification, target prioritization, and precision metabolic medicine.

Pooled CRISPR screening combined with single-cell RNA sequencing (scRNA-seq) has emerged as a powerful strategy for dissecting gene function and reconstructing gene regulatory networks (GRNs) in complex biological systems. This approach enables high-throughput, parallel perturbation of multiple genes while providing transcriptome-wide readouts at single-cell resolution, overcoming many limitations of traditional arrayed screens. However, its broader application remains limited by technical challenges, including variable perturbation efficiency and difficulties in accurately identifying perturbed cells. In this study, we adapted and applied a modified CRISPR droplet sequencing (CROP-seq) protocol using CRISPR interference (CRISPRi) in K562 cells to knockdown six transcription factors (TFs): LMO2, TCF3, LDB1, MYB, GATA2, and RUNX1. Our modified approach, which allows direct capture of sgRNAs from the cDNA library without a separate enrichment step, significantly improved sgRNA assignment per cell. We successfully achieved reproducible knockdown of three TFs (MYB, GATA2, and LMO2), captured the impact of these perturbations on the TF target genes, and enabled us to reconstruct their GRNs and identify key regulons and transcriptional targets. These networks revealed both previously established (such as LMO2 GATA2 interaction) and novel regulatory interactions, which we independently validated, providing new insights into hematopoietic transcriptional control. To assess the efficiency of CRISPRi based pooled perturbation, we additionally analyzed publicly available pertrub-seq CRISPRi datasets and found that only ∼40–50% of targeted genes led to effective knockdown, underscoring the variability in perturbation efficiency across experiments. Together, our results demonstrate both the potential and the current technical limitations of pooled CRISPRi-based single-cell screens. While this integrated approach holds great promise for high-resolution functional genomics, further optimization and standardized benchmarking are essential to improve its reliability, scalability, and reproducibility.

Abstract Muscle regeneration is governed by a complex interplay between immune cells and satellite cells (muscle progenitors), orchestrated by signaling molecules of the TGF-β superfamily. Among these, the role of GDF11 activity in skeletal muscle remains contentious, with conflicting evidence suggesting both stimulatory and inhibitory effects. This functional divergence may emerge from the combinatorial activities of its shared type I receptors and context-dependent activation of downstream SMADs. To dissect the role of GDF11 in skeletal myogenesis, we employed a combination of biochemical stimulation and CRISPR-based genetic approaches in chicken or human myoblasts. Analysis of cell proliferation, differentiation, adhesion, and migration revealed that GDF11 does not affect myoblast proliferation or adhesion, but strongly inhibits myotube differentiation and myoblast migration. Furthermore, loss of ACVR1B (ALK4) strongly delays myoblast differentiation, and impairs cell adhesion and migration on laminin-111 (LM111), a known ligand of the integrin VLA-6. Notably, flow cytometry phenotyping demonstrated that ACVR1B -deficient myoblasts exhibit reduced surface levels of the integrin α6 subunit (CD49f) compared to wild-type cells. Together, our findings suggest a GDF11-independent ALK4/VLA6/LM111 axis governing skeletal myoblast adhesion and fusion. Knowledge of these receptor interactions is critical for understanding GDF11’s paradoxical role in muscle cell biology and may inform novel therapeutic strategies to counteract skeletal muscle degeneration and age-related decline.

Fungal pathogens represent a major constraint to global agricultural productivity, causing a wide range of plant diseases that severely affect staple crops such as cereals, legumes, and vegetables. These infections result in substantial yield losses, deterioration of grain and produce quality, and significant economic impacts across the entire agri-food sector. Among phytopathogens, fungi are considered the most destructive, causing a wide range of diseases such as powdery mildew, rusts, fusarium head blight, smut, leaf spot, rots, late blight, and other fungal pathogens. Traditional plant protection methods do not always provide long-term effectiveness and environmental safety, which requires the introduction of innovative approaches to creating sustainable varieties. CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats) technology opens up new opportunities for targeted genome editing, allowing the modification or silencing of susceptibility genes and thus increasing plant resistance to fungal infections. This review presents current achievements and prospects for the application of CRISPR-Cas technology to increase the resistance of major agricultural crops to fungal diseases. The implementation of these approaches contributes to the creation of highly productive and resistant varieties, which is crucial for ensuring food security in the context of climate change.

Next-generation drug discovery and functional genomics require rapid, unbiased single-cell profiling at scale—demands that exceed the limited speed, throughput, and labor-intensive labeling constraints of conventional high-content image-based screening. We introduce spinning arrayed disk (SpAD), a high-throughput, label-free imaging platform for live-cell imaging that integrates continuous circular scanning, ultrafast quantitative phase imaging (QPI), and a novel circular array of 96 culture chambers. SpAD achieves an order-of-magnitude reduction in imaging time compared to traditional fluorescence-based workflows, while remaining compatible with standard cell culture workflows. By extracting rich biophysical features using intrinsic morphological (InMorph) profiling and machine learning, SpAD enables sensitive, large-scale screening of drug responses and CRISPR gene knockouts without labeling. Critically, label-free biophysical readouts from SpAD reveal mechanism-linked changes in mass, refractive index, subcellular textures, and light scattering that fluorescent labels often obscure. SpAD thereby resolves subtle phenotypes and heterogeneous subpopulations with high reproducibility, providing a robust, scalable foundation for precision cellular morphological assays.

The endogenous opioid system is a powerful modulator of motivation and affect. The dorsal raphe nucleus (DRN) in the midbrain has been established as an important site of opioid action and is an integral hub in behavioral modulation. To investigate the functional significance of DRN opioid signaling in aversive and appetitive behaviors we disrupted preproenkephalin (Penk) in DRN using CRISPR-Cas9 technology in Penk-Cre mice. We found that CRISPR mediated knockdown of enkephalin peptide in the DRN (DRN Penk ) enhanced inflammation-induced mechanical sensitivity and odor avoidance. Additionally, loss of DRN Penk diminished sucrose preference and engagement with a novel social stimulus. To further characterize the opioid system within the DRN, we performed Hiplex in situ hybridization of 12 genes in the same tissue. This revealed that DRN Penk is largely separate from DRN serotonin cells and is instead distributed on glutamatergic and GABAergic cells. However, subtype-specific knockdown of DRN Penk from glutamatergic and GABAergic cells did not replicate the behavioral effects of general DRN Penk knockdown. This suggests that these neurons represent a novel population that mediate motivated behaviors distinctly from canonical DRN mechanisms.

Abstract Gene expression during cellular differentiation is coordinated by combinatorial interactions between transcription factors (TFs) and cofactors at promoters and enhancers. The “master TF” GATA1 coordinates gene transcription in a subset of hematopoietic lineages, including erythroid, megakaryocytic, mast, and eosinophil, while repressing the development of other blood lineages. However, the specific cofactors required for GATA1-activated gene expression during hematopoiesis are incompletely defined. We identified the cofactor KMT2D, an H3K4 methyltransferase that collaborates with H3K27 acetyltransferases to activate transcription, in an unbiased CRISPR/Cas9 screen for epigenetic regulators of erythropoiesis. Loss of KMT2D in human erythroid precursors caused developmental arrest with impaired expression of numerous erythroid genes. Mechanistically, KMT2D colocalized with GATA1 on more than one thousand erythroid enhancers associated with over two hundred erythroid genes. In general, co-occupancy of GATA1 and KMT2D at erythroid enhancers was associated with stronger transcriptional activity than occupancy by GATA1 alone. Acute depletion of KMT2D in erythroid precursors caused rapid reductions of H3K4me1 and H3K27ac on a subset of GATA1-bound enhancers and impaired the expression of canonical erythroid genes, including ZFPM1, SLC4A1 , and EPOR . Moreover, acute depletion of GATA1 or KMT2D individually caused downregulation of overlapping gene sets. Thus, KMT2D controls erythropoiesis by selectively activating GATA1-dependent erythroid enhancers. Our studies identify KMT2D as a novel cofactor for transcriptional activation by GATA1 during erythropoiesis. More generally, our findings demonstrate how a lineage-specific TF cooperates with a ubiquitous epigenic regulator to drive lineage-specific gene expression during cellular differentiation.

Deciphering the functionality of the noncoding genome which includes important cis -regulatory elements (CREs) and transcribed noncoding RNA genes remains technically challenging. Here, using massively parallel genetic screening, we systematically benchmark the performance of five representative loss-of-function perturbation tools, including single guide RNA (gRNA) mediated SpCas9 cleavage or CRISPR interference, and paired gRNA (pgRNA) involved dual-SpCas9, Big Papi (paired SpCas9 and SaCas9) or dual-enAsCas12a fragment deletion methods, in decoding the roles of the noncoding genome. For targeting CREs such as enhancer, dual-SpCas9 outperforms other methods with superior efficiency of destroying functional genomic regions. For perturbing noncoding RNA genes, in addition to dual-SpCas9, other RNA-targeting methods such as RNA interference are recommended to discriminate transcript-dependent or -independent roles. A deep learning model DeepDC with associated web server is built to facilitate optimal dual-SpCas9 pgRNA design for efficiently deleting a genomic fragment. Together, our work provides practical guidance on selecting appropriate loss-of-function tools to resolve the functional complexity of the noncoding genome.

ABSTRACT Transcriptional regulation is mediated by enhancers, yet how genetic perturbations alter enhancer activity and gene expression remains poorly understood. We developed UDI-UMI-STARR-seq, which integrates dual indexes and unique molecular identifiers, and combined it with RNA-seq to profile the effects of perturbations on enhancer activity and target gene expression. We applied this approach to a library of 253,632 fragments representing 46,142 cell type–specific candidate enhancers and assessed the impact of CRISPR/Cas9-mediated deletion of six transcription factors (or TFs; ATF2, CTCF, FOXA1, LEF1, TCF7L2, and SCRT1) with diverse regulatory roles. Across knockout lines, we identified responsive enhancers that were either repressed or induced, often through motifs such as the p53 family of TFs. Enhancer–gene mapping revealed TF-specific programs, including repression of Wnt/p53 targets with ATF2 or LEF1 loss, downregulation of the FIRRE locus with CTCF loss, and compensatory upregulation of RNA polymerase II regulators following FOXA1 depletion. A deep learning model trained on enhancer sequences recapitulated core principles of enhancer grammar, including cooperative motif syntax and the influence of flanking sequence context. Applying this framework to the neurodevelopmental disorder-associated 16p12.1 deletion identified responsive enhancers linked to genes involved in axon guidance, synaptic plasticity, and translational control, providing a scalable readout of enhancer dynamics generalizable to genetic mutations.

Abstract Some childhood cancers can arise in utero and then regress at birth, but the cues that permit malignant proliferation in utero as opposed to postnatal life are often unclear. Transient abnormal myelopoiesis (TAM) is a human fetal liver leukemia driven by GATA1s mutations and a rare exemplar of a spontaneously resolving cancer when blood formation shifts from liver to bone marrow (BM) during development. Here we show that the cytokine receptor CSF2RB is aberrantly upregulated in TAM cells because it is a GATA2 target that escapes repression by GATA1s. Pathologically expressed CSF2RB unexpectedly interacts with the thrombopoietin receptor MPL to prolong JAK-STAT signaling by fetal-liver produced THPO, driving GATA1s-mutant cell expansion. TAM can transform into myeloid leukemia of Down syndrome (ML-DS) upon acquisition of additional mutations. We further show that the ML-DS driver CSF2RB A455D forces MPL dimerization resulting in constitutive JAK-STAT activation, bypassing THPO dependence in the fetal-liver niche, thereby enabling proliferation in the BM. Conversely, base-editing reversion of another ML-DS JAK-STAT-activating mutation, JAK3 A572V, restores THPO dependence. These results identify a cytokine gate that developmentally restricts GATA1s oncogenic competence, reconciling why TAM expands in the fetal liver yet resolves after birth, revealing a niche-specific, therapeutically targetable dependency.

Abstract MHC-II molecules are traditionally restricted to professional antigen-presenting cells (pAPCs), but increasing evidence highlights their expression in cancer cells, where they are associated with enhanced immune infiltration and improved clinical outcomes. However, the mechanisms governing cancer cell-intrinsic MHC-II expression remain poorly understood. Here, through genome-wide CRISPR-Cas9 screening in human melanoma cells, we identify the aryl hydrocarbon receptor (AHR) and its dimerization partner (ARNT) as critical, ligand-responsive regulators of MHC-II expression. Our analyses reveal that AHR–ARNT promotes transcription of the MHC-II transactivator CIITA through direct binding to its promoter II (pII), independently of IFN-γ signaling. Clinically, an AHR–ARNT loss-of-function signature correlates with reduced immune infiltration, poor response to immunotherapy, and inferior survival across cancer types. Together, our findings uncover a previously unrecognized, tumor-intrinsic regulatory axis of MHC-II expression and suggest that targeting the AHR–ARNT pathway may enhance tumor immunogenicity and improve responses to immunotherapy.

Predatory bacteria are abundant in soil, but their diversity and functions remain not fully understood, especially in subarctic regions. Here, we report strain 1-FT3.2, a predatory bacterium obtained from peatland soil in Northern Finland (Pallas, 68 °N). The bacterium was cultivated on Mucilaginibacter cryoferens FT3.2 as prey. Although a pure culture of strain 1-FT3.2 was not obtained, its draft genome was assembled from sequencing reads derived from the co-culture with its prey. The draft genome of 1-FT3.2 is 7.2 Mb in length and 81% complete. Genome analyses suggested that 1-FT3.2 belongs to the family Polyangiaceae (phylum Myxococcota ), which comprises predatory bacteria. The genome annotation revealed (near-)complete metabolic modules of central carbon metabolism and aerobic respiration. Two proviral regions were predicted in the draft genome, both putatively representing tailed phages of the class Caudoviricetes. Several CRISPR-Cas system proteins were also identified. The draft genome sequence could be used in future comparative studies assessing the diversity of predatory bacteria in northern soils or other environments.