Abstract Grain length is a critical agronomic trait that directly determines rice yield. In this study, we identified OsvWA36, a von Willebrand factor A (VWA) domain protein containing intrinsically disordered regions (IDRs). We showed that it is a novel positive regulator of grain length and that its function is achieved through liquid-liquid phase separation (LLPS) and subsequent modulation of cell wall remodeling.OsvWA36 was discovered through proteomic screening, and its abundance was positively correlated with grain length. It preferentially accumulated in developing panicles and formed liquid-like condensates via IDR-mediated LLPS, as indicated by in vitro/in vivo assays and fluorescence recovery after photobleaching analysis. CRISPR-Cas9-generated osvwa36 mutants developed shorter grains due to reductions in glume cell length and the aberrant accumulation of lignin. Transcriptomic and qRT-PCR analyses revealed that deficiency in OsvWA36 suppressed the expression of genes associated with cell wall dynamics, including those involved in cellulose synthesis ( OsCESA4, OsCESA7 , and OsCSLE1 ), pectin metabolism ( OsPME68, OsPME1 ), and lignin modification ( OsCAD2, OsMYB58 , and OsExo70H3 ). Genetic complementation restored the wild-type phenotype, whereas overexpression of OsvWA36 further elongated grains. Deletion of the IDR domain abolished LLPS and resulted in short grains, phenocopying the osvwa36 mutants and underscoring the functional necessity of phase separation. Furthermore, haplotype analysis revealed that natural variation in OsvWA36 was correlated with grain length diversity in rice cultivars.In conclusion, our findings indicate that OsvWA36 regulates grain length by orchestrating cell wall remodeling through IDR-mediated LLPS and could be a useful target for molecular breeding strategies aimed at improving yield.

High-fat diet (HFD)-induced obesity remains a significant global health challenge. In this study, we show that a global knock-in CRISPR mouse with the Kir2.1 L222I single-point mutation exhibits remarkable resistance to HFD-induced obesity. We identify palmitic acid (PA), a prevalent long-chain fatty acid in obesity, as a novel negative regulator of Kir2.1. Kir2.1 L222I previously shown to protect against cholesterol-mediated inhibition of Kir2.1, also confers protection against PA-induced suppression. Moreover, PA-induced suppression of Kir2.1 results in a significant loss of flow-induced vasodilation (FIV), while the L222I mutation exerts a protective effect. Notably, Kir2.1 L222I mice display significant protection against HFD-induced weight gain and adiposity independent of caloric intake. Specifically, the mutant mice show increased lean mass and decreased fat mass, specifically in both visceral and subcutaneous white adipose tissue (WAT) and intrascapular brown adipose tissue (BAT). Importantly, visceral-to-subcutaneous white adipose ratios decrease while BAT/WAT tissue ratios increase, suggesting a metabolically favorable fat distribution. This protection correlates with enhanced physical activity and increased energy expenditure. Metabolomic analysis reveals elevated TCA cycle metabolites in adipose tissue of Kir2.1 L222I mice, consistent with their enhanced energy expenditure. These findings highlight Kir2.1 channels as potential therapeutic targets for obesity and related metabolic disorders.

Hypoxia sensing via the Cys/Arg branch of the N-degron pathway (Cys-NDP) is central for flooding responses in plants, yet how evolutionary and ecological factors have shaped the core oxygen sensing mechanism remains poorly understood. Leveraging the publication of multiple angiosperm genomes, we systematically analysed known Cys-NDP components in 55 angiosperms spanning aquatic, epiphytic, xerophytic, and mesophytic lineages. We also complemented this survey with hypoxia profiling and transcriptomic analyses in a selected panel of plants. This comparative effort revealed variation in Cys-NDP components, with Plant Cysteine Oxidases (PCOs) and group VII Ethylene Response Factors (ERFVIIs) emerging as major sources of diversification. Aquatic monocots displayed complete loss of A-type PCOs and dramatic expansion of a novel clade of ERFVIIs (HRE aqua ), frequently accompanied by loss or modification of the Cys-degron, uncoupling them from oxygen-dependent turnover. By contrast, xerophytes and epiphytes retained core Cys-NDP elements but showed shared hypoxia-induced gene expression, suggesting endogenous developmental or metabolic pressures for pathway conservation in habitats with limited flooding risk. Across all species, we identified a conserved transcriptional core of 11 orthogroups, including fermentation enzymes and regulatory factors, highlighting the early recruitment of these genes to hypoxia responses. Functional assays confirmed contributions of conserved MYB and LBD transcription factors to hypoxia tolerance in Arabidopsis . Together, our results demonstrate that both habitat and anatomy influence the evolution and deployment of oxygen-sensing networks in angiosperms. While persistent submergence promoted diversification of ERFVIIs and PCOs, retention of the core pathway across lineages points to fundamental roles in coping with endogenous oxygen gradients and fluctuations.

Background The PIWI-interacting RNA (piRNA) pathway is the primary defense against the deleterious activity of transposable elements (TEs), a role classically assigned to the germline. We recently discovered that the retrotransposon Copia is a negative regulator of synaptogenesis at the Drosophila larval neuromuscular junction (LNMJ) [1]. Here, we investigated whether the piRNA pathway regulates Copia in this somatic context. Methods Analysis of existing sequencing data revealed the expression of piRNA pathway components in somatic tissues [2]. We focused on Aubergine ( aub ), a core PIWI-clade Argonaute. We utilized CRISPR generated aub reporter lines and confocal microscopy to confirm the enrichment of AUB at the LNMJ and next generation sequencing coupled with digital PCR to validate the upregulation of TEs in aub knockdown larvae and adult tissues. Results Data from genetic reporters and antibody staining show that AUB is expressed and localized to the LNMJ. Tissue-specific knockdown of aub at the LNMJ resulted in increased TE expression, including Copia . In contrast to the synaptic overgrowth seen with Copia depletion [1], aub reduction caused a decrease in synapse number and impaired motor function and lifespan. These phenotypes are consistent with the upregulation of Copia , a negative regulator of synapse growth. Conclusions Our findings demonstrate that AUB functions somatically at the LNMJ to repress TEs, thereby ensuring proper neuromuscular development and function. This work establishes a physiological role for the piRNA pathway in a somatic tissue, linking TE repression to neuromuscular development.

CRISPR-based therapeutics rely on guide RNAs (gRNAs) and the Cas9 endonuclease for precise gene editing. Ensuring gRNA purity and base-level sequence integrity is essential for clinical translation. While industry-standard practice relies on liquid chromatography-high-resolution mass spectrometry to assess oligonucleotide identity and purity, more recent FDA guidance recommends complementary base-by-base sequence analysis (FDA CBER Webinar, 2024). In this study, we evaluated next-generation sequencing (NGS) strategies for characterizing chemically synthesized gRNAs. We found that the widely used SMARTer assay, while capable of producing sequenceable libraries, introduced substantial artifacts during library preparation. These included truncated scaffold species at oligo(A) stretches in the scaffold region and 5′(n-1) deletions within the spacer sequence. Although absent in the original gRNA, these artifacts accounted for over 10% of the sequencing reads, creating the false appearance of impurities. Through experimental and computational approaches, we traced these artifacts to mispriming by template-switching oligonucleotides (TSOs). Importantly, these artifacts occur during sequencing, and although they do not reflect real gRNA impurities, they compromise assay accuracy and can obscure true sequence impurities. To overcome these limitations, we developed FUSS-seq (Full-length Uncoupled Second-strand Synthesis followed by sequencing), a novel assay that integrates principles from 5′ RACE with a modified TSO bearing a 3′ polymerase-blocking moiety. FUSS-seq markedly reduced artifacts and increased full-length gRNA recovery, providing a more accurate and lower-bias method for gRNA purity assessment. This approach supports improved Chemistry Manufacturing and Controls (CMC) characterization of gRNAs and strengthens the analytical toolkit needed for reliable CRISPR-based therapeutic development.

Predicting cellular responses to genetic perturbations is critical for advancing our understanding of gene regulation. While single-cell CRISPR perturbation assays such as Perturb-seq provide direct measurements of gene function, the scale of these experiments is limited by cost and feasibility. This motivates the development of computational approaches that can accurately infer responses to unmeasured perturbations from related experimental data. We introduce dbDiffusion, a generative framework that integrates diffusion models with classifier-free guidance derived from perturbation information, operating in latent space through a variational autoencoder (VAE). Diffusion models are probabilistic generative models that approximate data distributions by reversing a Markovian diffusion process, progressively denoising Gaussian noise into structured outputs. By exploiting biological similarities in gene expression profiles and relationships among perturbations, dbDiffusion enables the conditional generation of gene expressions for previously unobserved perturbations. In contrast to competing approaches, dbDiffusion does not rely on LLM or foundation models, which have been found to yield unsatisfactory results. Rather, it leverages embeddings derived from measured perturbations to generalize to unseen pertur-bations, effectively transferring information across related experimental conditions. In benchmarking against state-of-the-art methods on Perturb-seq datasets, dbDiffusion demonstrates superior accuracy in predicting perturbation responses. A methodological innovation of dbDiffusion is the integration of prediction-powered inference, which corrects for biases inherent in generative models and enables statistically rigorous downstream tasks, including identification of differentially expressed genes. By combining deep generative modeling with principled inference, dbDiffusion establishes a scalable computational framework for predicting and analyzing transcriptomic perturbation responses, significantly extending the utility of Perturb-seq experiments.

The gut microbiota has emerged as a critical immune-metabolic interface, orchestrating a complex network of interactions that extend well beyond digestion. This highly diverse community of bacteria, viruses, archaea, and eukaryotic microbes modulates host immunometabolism, metabolic reprogramming, and systemic inflammatory responses, thereby shaping human health and disease trajectories. Dysbiosis, or disruption of microbial homeostasis, has been implicated in inflammatory bowel disease, cardiometabolic disorders, neurodegeneration, dermatological conditions, and tumorigenesis. Through the biosynthesis of short-chain fatty acids (SCFAs), bile acid derivatives, tryptophan metabolites, and microbial-derived indoles, the gut microbiota regulates epigenetic programming, barrier integrity, and host–microbe cross-talk, thereby influencing disease onset and progression. In oncology, specific microbial taxa and oncomicrobiotics (cancer-modulating microbes) are increasingly recognized as key determinants of immune checkpoint inhibitor (ICI) responsiveness, chemotherapeutic efficacy, and resistance mechanisms. Microbiota-targeted strategies such as fecal microbiota transplantation (FMT), precision probiotics, prebiotics, synbiotics, and engineered microbial consortia are being explored to recalibrate microbial networks and enhance therapeutic outcomes. At the systems level, the integration of multi-omics platforms (metagenomics, metabolomics, transcriptomics, and proteomics) combined with network analysis and machine learning-based predictive modeling is advancing personalized medicine by linking microbial signatures to clinical phenotypes. Despite remarkable progress, challenges remain, including the standardization of microbiome therapeutics, longitudinal monitoring of host–microbe interactions, and the establishment of robust ethical and regulatory frameworks for clinical translation. Future directions should prioritize understanding the causal mechanisms of microbial metabolites in immunometabolic regulation, exploring microbial niche engineering, and developing precision microbiome editing technologies (CRISPR, synthetic biology).

ABSTRACT Background Duchenne muscular dystrophy (DMD) is a lethal pediatric degenerative muscle disease for which there is no cure. Robust preclinical models that recapitulate major clinical features of DMD are required to investigate efficacy of potential DMD therapeutics. Rat models of DMD have emerged as promising small animal models to accomplish this; however, there have been no comprehensive studies investigating the functional skeletal muscle decrements associated with the modeling of DMD in rats. Methods CRISPR/Cas9 gene editing was used to generate a dystrophin-deficient Sprague-Dawley muscular dystrophy rat (MDR). Biochemical and immunofluorescent analyses were performed to confirm loss of dystrophin in striated muscles of this rat model. In situ and ex vivo muscle function was assessed in wild-type (WT) and MDR muscles at 3, 6, and 12 months of age, followed by histopathological analyses. Results MDR muscle tissues exhibited loss of full-length dystrophin and reduced content of other dystrophin glycoprotein complex members. MDR extensor digitorum longus (EDL) muscles and diaphragms displayed pronounced and progressive muscle weakness beginning at 3 months of age, compared to WT littermates. EDLs also exhibit susceptibility to eccentric contraction-induced damage. Functional deficits in soleus muscles were less severe and were associated with a right shift in force-frequency relationship and a muscle fiber-type shift. MDR muscles display progressive histopathology including degenerative lesions, fibrosis, regenerative foci, and modest adipose deposition. Conclusions MDR is a preclinical model of DMD that exhibits many translational features of the human disease, including a large dynamic range of muscle decrements, that has high utility for the evaluation of potential therapeutics for DMD.

The large size of CRISPR-Cas enzymes limits their delivery for therapeutic applications. Cas12j nucleases offers hypercompact alternative but show moderate editing efficiency. To overcome this limitation, we identified eight novel Cas12j orthologues (Cas12j-11 to Cas12j-18) from viral metagenomes. All showed low editing activity in mammalian cells. We engineered T5 exonuclease-Cas12j fusions (T5Exo-Cas12j), two of which, T5Exo-Cas12j-12, and -18 exhibited up to 42% editing in HEK293T and 9% in K-562 cells, outperforming wild-type Cas12j counterparts and comparable to LbCas12a. Intriguingly, robust in cellula editing in both HEK293T and K-562 cells was strictly dependent on the presence of 5′-TAC trinucleotides within the target DNA sequence. Furthermore, we fused the Cas12j orthologues with the TadA8e deaminase and developed base editors, termed Be-(d)Cas12j. Among these, Be-(d)Cas12j-13 demonstrated efficient A-to-G base conversion in mammalian cells. This study expands the CRISPR toolbox by characterizing and engineering novel Cas12j orthologues into compact, high-efficiency genome editors.

Summary Diatoms are a highly diverse group of phytoplankton that have a large impact on global primary production and carbon sequestration in the ocean 1,2 . However, they are evolutionarily divergent from model phototrophs of the green lineage, and limited screening tools have hampered discovery of unique diatom biology. To address this challenge, we developed a genome-wide CRISPR/Cas9 screen in the model marine diatom, Phaeodactylum tricornutum. The screen was applied to identify genes required for survival in different light regimes, including both high light and fluctuating light. We identified a broad set of uncharacterized genes, providing the foundation for mechanistic studies of diatom adaptation to dynamic light. Among these genes, we demonstrated that the red lineage-exclusive gene STROBE1 is a new potentiator of cyclic electron flow (CEF) required for CEF to generate a trans-thylakoid proton gradient 3,4 . As dynamic light conditions are common in marine environments, STROBE1 and other genes identified in this screen may contribute to the broad ecological success of diatoms 5,6 . This genome-wide genetic screen in P. tricornutum will accelerate the unbiased discovery of novel gene functions in these ecologically important organisms.

ABSTRACT Eukaryotic genomes generate a plethora of polyadenylated (pA + ) RNAs 1,2 , that are packaged into ribonucleoprotein particles (RNPs). To ensure faithful gene expression, functional pA + RNPs, including protein-coding RNPs, are exported to the cytoplasm, while transcripts within non-functional pA + RNPs are degraded in the nucleus 1–4 . How cells distinguish these opposing fates remains unknown. The DExD-box ATPase UAP56/DDX39B is a central component of functional pA + RNPs, promoting their docking to the nuclear pore complex (NPC)-anchored ‘transcription and export complex 2 (TREX-2)’ (ref. 5,6 ), which triggers transcript release from UAP56 to facilitate export (ref. 7,8 ). Here, we uncover that the ‘Poly(A) tail exosome targeting (PAXT)’ connection 9 harbors its own TREX-2-like module, which releases pA + RNAs from UAP56 for decay by the nuclear exosome. The core of this module consists of a LENG8-PCID2-SEM1 (LENG8-PS) trimer, which we show is structurally and functionally equivalent to the central GANP-PCID2-SEM1 (GANP-PS) trimer of TREX-2. Mutagenesis and transcriptomic data demonstrate that the nuclear fate of pA + RNPs is governed by the contending actions of nucleoplasmic PAXT and NPC-associated TREX-2, which interpret RNA-bound UAP56 as a signal for RNA decay or export, respectively. As RNA targets of PAXT are generally short and intron-poor, we propose an overall model for pA + RNP fate determination, whereby the distinct sub-nuclear localizations of PAXT and TREX-2 govern the degradation of short non-functional pA + RNAs while allowing export of their longer and functional counterparts.

Abstract Background: Safety and specificity remain major challenges in viral gene therapy for cancer and tissue repair. The Crucible Virus integrates replication deficient lentivirus, herpes simplex virus type one, and influenza A vectors into a single insulated chassis that enforces multi-input promoter gating and multi-tier kill switches to achieve conditional activation and enhanced safety. Methods: Vectors incorporate heat and cytokine responsive promoters HSP70 and NF kappa B, cHS4 insulators, and unique DNA barcodes. In vitro assays quantified promoter induction, basal leak, and engagement of CRISPR Cas nine and protease cleavable degron kill switches. Seven orthotopic and xenograft tumor models received sequential or combined dosing via intravenous and intralesional routes with assessments of biodistribution, immunogenicity, survival, and tumor volume. Results: The HSP70 and NF kappa B promoters achieved up to 168-fold induction with less than 0.2 percent basal leak. Off target CRISPR excision removed more than 90 percent of the payload within ninety minutes, and degron clearance reduced secreted effectors by half in under one hour. Median survival improved by thirty to sixty percent with probability value less than 0.01, and tumor volumes shrank by forty to fifty two percent compared to controls. Vector genomes cleared to below five thousand copies per microgram DNA by day twenty-eight, with off target activation under 0.5 percent and manageable antibody titers. Conclusions: The Crucible Virus delivers robust antitumor efficacy and regenerative potential under rigorous safety controls. Its modular traceable design supports scalable manufacturing and an investigational new drug ready profile for precision oncology and regenerative medicine applications.

The recent development of long-read sequencing has made it possible to catalog variable number tandem repeats (VNTRs) in the human genome. However, little is known about their functional consequences. Here, we characterized the effect of TRACT, a VNTR that is unique to humans and that has sequence variants linked to risk for bipolar disorder and schizophrenia. By adding or removing this VNTR in both mouse models and human neural organoids, we find that TRACT, which is intronic to the L-type voltage-gated calcium channel gene CACNA1C , increases intracellular calcium after neuronal stimulation and leads to widespread changes in activity-dependent transcription programs in neurons. TRACT-dependent changes are enriched for genes associated with synapse formation and plasticity, and partially recapitulate evolutionary changes in activity-dependent transcription between species. These findings demonstrate that a single, human-specific, non-coding element can strongly affect the neuronal response to stimulation, and motivate the study of VNTRs as a genetic source of phenotypic variation in both evolution and disease.

The human aryl hydrocarbon receptor (AHR) integrates chemical signals derived from the environment, gut microbes, and endogenous sources to regulate processes ranging from intestinal barrier integrity to xenobiotic detoxification. Despite strong evidence that dysregulation of AHR signaling is a causal factor in metabolic and autoimmune disorders, we currently lack a comprehensive understanding of the factors that regulate AHR activity in human cells. Here, we use genome-scale CRISPR screening to systematically identify regulators of AHR signaling in hepatocytes. The resulting datasets recapitulate the core AHR signaling pathway and identify a large network of regulators. Many of these factors have roles beyond AHR signaling, reflecting that AHR signaling is deeply integrated into human cell biology. We further dissect this network to reveal novel modes of regulation of AHR expression, protein levels, and signaling. For example, we find that the E3 ubiquitin ligase UBR5 sustains AHR signaling by counteracting degradation of ligand-bound AHR. Finally, we identify components of the AHR regulatory network that are specific to cell types and ligands as potential nodes to manipulate AHR signaling in a targeted manner for therapeutic benefit. Overall, our results define the regulatory network that underpins AHR activation, with implications for our understanding of host-microbe interactions and integrative chemosensation and the etiology of metabolic and inflammatory disorders.

FACS-based CRISPR screening has emerged as a potent tool for dissecting the genetic networks that regulate cell-surface glycosylation. However, existing protocols are tedious and poorly suited to many cell models. We developed a lectin-based magnetic-activated cell sorting platform (Lec-MACS) that enables rapid identification of genes controlling expression of specific cell-surface glycans. Lec-MACS is faster and easier to perform than FACS-based screening while producing data of similar quality. We subsequently applied Lec-MACS to produce a genomic atlas of genes regulating breast cancer hypersialylation. This method will dramatically expand the scope and throughput of genetic screens targeted at cell-surface glycans.

Tephritidae insect pests account for extensive crop damage and yield losses globally. Modern, sustainable pest management approaches are species-specific and, therefore, high-quality genome assemblies are required for their application. Here, we present chromosome-level assemblies for five members of the Tephritidae family: Anastrepha fraterculus, Anastrepha ludens, Bactrocera dorsalis, Bactrocera zonata and Zeugodacus cucurbitae . The assemblies used long read sequencing polished with short read sequencing and scaffolded using Hi-C (chromatin conformation capture) sequencing. Prior to scaffolding the assembly deduplication was performed to separate a primary assembly and an alternate assembly, and each was then scaffolded independently. The scaffolded assemblies reached N50 length in the range of 60Mb to 120Mb. The scaffolded assemblies were verified with BUSCO and completeness was in the range 97% to 98.5% and had very low duplicated, fragmented and missing orthologs.

Background and Aims Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy characterized by diagnosis at advanced stages, limited therapeutic options, and frequent resistance to therapies. Although oncogenic KRAS mutations are central drivers of PDAC, alternative pathways indisputably contribute to its tumorigenesis and progression. RhoV, a member of the Rho family of small GTPases, has been implicated in tumor development in other cancer types, such as breast cancer and lung adenocarcinoma; however, its role in PDAC remains unclear. Methods In this study, we investigated the expression and functional impact of RhoV on PDAC. Analysis of publicly available datasets and immunohistochemical profiling of 114 PDAC patient specimens were used to evaluate the expression of RhoV in PDAC and its prognostic impact. Overexpression and CRISPR-Cas9-mediated knockout of RhoV were established in three pancreatic cancer cell lines. Functional analyses, such as cell proliferation, migration, invasion, colony formation, spheroid growth, and mouse xenograft, were used to evaluate the role of RhoV in PDAC cells. Results RhoV overexpression was associated with reduced overall and recurrence-free survival in public datasets and our own patient cohort. Functional assays demonstrated that RhoV overexpression promoted PDAC cell proliferation, colony formation, and spheroid growth, whereas knockout of RhoV suppressed these changes. Moreover, RhoV enhanced PDAC cell migration and invasion in vitro , accompanied by downregulation of E-cadherin and upregulation of N-cadherin and vimentin, indicating induction of epithelial–mesenchymal transition. Mechanistically, RhoV overexpression activated key MAPK pathway components, including phosphorylation of ERK, JNK, and p38. In vivo , xenograft models confirmed that RhoV drives tumor growth and increases tumor burden. Conclusion These results establish RhoV as a novel oncogenic factor in PDAC progression and highlight its potential as a biomarker and therapeutic target, warranting further investigation into combinatorial targeting strategies to overcome KRAS inhibitor resistance.

Background Musculoskeletal diseases are a leading cause of global disability and healthcare burden, yet traditional orthopaedic procedures often fail to address molecular drivers of degenerative and genetic conditions. CRISPR-Cas Systems, a precise and programmable genome-editing technology, show promise in preclinical musculoskeletal models, ranging from gene knockouts in osteoarthritis to mutation correction in skeletal dysplasia. Despite growing interest, no comprehensive synthesis exists to map how CRISPR-Cas Systems are being applied in orthopaedic research. This scoping review aims to fill that gap. Methods This protocol will be registered with the Open Science Framework and follows PRISMA-ScR guidelines and the Arksey & O’Malley framework. MEDLINE, Embase, Scopus, Web of Science, and grey literature sources from 2005 onward will be searched. Inclusion criteria encompass original research using CRISPR-Cas Systems in human, animal, or in vitro musculoskeletal models. Two reviewers will independently screen titles, abstracts, and full texts using Covidence software. Data extraction will be standardized and performed in duplicate. Extracted variables include study design, model system, target tissue, gene-editing approach, delivery system, and reported outcomes. Results will be synthesized descriptively and thematically. Expected Results We anticipate mapping the evolution and diversity of CRISPR-Cas Systems applications across musculoskeletal tissues (bone, cartilage, tendon, muscle), highlighting domains such as tissue regeneration, gene correction, and disease modeling. Delivery strategies (viral vectors, nanoparticles) and translational challenges, including off-target effects and delivery barriers, will be summarized. Conclusions This will be the first scoping review to systematically characterize the role of CRISPR-Cas Systems in musculoskeletal medicine. Findings will inform researchers, clinicians, and policymakers, helping to guide future translational research and accelerating the integration of gene editing into clinical practice.

Advances in functional genomic technology, notably CRISPR using Cas9 or Cas12, now allow for large-scale double perturbation screens in which pairs of genes are inactivated, allowing for the experimental detection of genetic interactions (GIs). However, as it is not possible to validate GIs in high-throughput, there is no gold standard dataset where true interactions are known. Hence, we constructed a Double-CRISPR Knockout Simulation (DKOsim), which allows users to reproducibly generate synthetic simulation data where the single gene fitness effect of each gene and the interaction of each gene pair can be specified by the investigator. We adapted Monte-Carlo randomization methods to extend single knockout simulation methods to double knockout designs, which simulate the gene-gene interactions between all possible combinations of the input genes. Using DKOsim, we generated simulated datasets that closely resemble real double knockout CRISPR datasets in terms of Log Fold Change (LFC), GI distribution, and replicate correlation. We further inferred optimal CRISPR library designs by systematically investigating critical experimental parameters including depth of coverage, guide efficiency, and the variance of initial guide distribution. This simulation scheme will help to identify optimal computational methods for GI detection and aid in the design of future dual knockout CRISPR screens. Author Summary We designed DKOsim to simulate CRISPR double knockout screens by modeling cell division behavior with both single knockout (SKO) and double knockout (DKO) constructs via Monte-Carlo randomization samplers. Running DKOsim at large scale, we identified the asymptotic tuning points that optimize genetic interaction (GI) identification performance by the delta-LFC (dLFC) method compared to the simulated truth. We show that DKOsim is tunable to approximate actual dual-CRISPR knockout screening data. Comparing replicate correlation from DKOsim with experimentally generated data, DKOsim can be tuned based on users’ desires to reproduce a similar level of randomness to that observed in variety CRISPR screening conditions.

ABSTRACT Insufficient protein intake, leading to malnutrition, is a major global health concern that compromises the immune system and increases susceptibility to diseases. In scenarios where protein availability is constrained, organisms may experience strong selection to efficiently allocate their resources between immunity vs other energy-demanding processes, such as reproduction, resulting in evolutionary trade-offs. Additionally, in many species, protein deficiency has a more significant impact on female reproduction than on males, potentially leading to pronounced sexually dimorphic trade-offs involving immunity and infection outcomes. However, there are no experiments to test these possibilities. In this work, we demonstrate that in Drosophila melanogaster populations selected for increased early-life reproduction under protein limitations, evolved virgin females indeed suffered a greater reduction in their resistance to the pathogen Providencia rettgeri and showed lower survival following infection than males, corroborating our expectations. However, mating resulted in a loss of sexually dimorphic infection outcomes, causing both sexes to exhibit nearly identical infection costs and reduced infection tolerance compared to their ancestral, unselected populations. Moreover, several immune components, including the Toll- and IMD-mediated inducible immune pathways, were either less upregulated or more downregulated in the selected flies, which may contribute to their heightened susceptibility to pathogens. Downregulation in key metabolic pathways and genes related to phagocytosis, melanisation and ROS-mediated defence after infection in selected flies can also be associated with their increased pathogen vulnerability. Taken together, our work thus reveals the reproductive status- and sex-specific plasticity of immune investments and post–infection health in response to evolutionary constraints under chronic protein deprivation.

The CRISPR-Cas system enables precise genome engineering of cell therapies. For allogeneic applications, multiplex editing is frequently required to improve efficacy, persistence, and safety. However, strategies involving multiple DNA double-strand breaks (DSBs) induce genotoxicity by provoking chromosomal aberrations. Base editors, which enable sequence changes without generating DSBs, are widely used for gene disruption, but their capacity for gene insertion remains unexplored. Here, we developed B ase e ditor-mediated k nock- i n ( BEKI ), a non-viral platform that allows targeted transgene insertion in parallel with multiplex gene disruption using a single base editor. Repurposing the Cas9 nickase domain of base editors generates paired nicks, inducing homology-directed repair (HDR). In human T cells, optimized guide RNA orientation and nick distance, together with HDR-enhancing modulators, enabled efficient transgene knock-in at the TRAC , CD3ζ, B2M, and CD3ε loci. Simultaneous base editing of multiple additional genes produced chimeric antigen receptor (CAR) T cells with increased cytokine secretion, drug resistance, and resistance to allo-rejection. Compared to multiplex editing with Cas9, BEKI markedly reduced chromosomal translocations. BEKI therefore provides a streamlined, scalable strategy for multiplex CAR T-cell engineering with a single enzyme, offering a safer route to clinical-grade manufacturing of off-the-shelf therapies for cancer and autoimmune diseases. Graphical Abstract

Abstract Acute Myeloid Leukemia (AML) remains challenging to treat, especially in cases with mutations in the BCL-6 co-repressor (BCOR), which are associated with poor prognosis and chemo-resistance. In this study, we reveal a synthetic lethal interaction between BCOR and dihydroorotate dehydrogenase (DHODH). We demonstrate that BCOR -deficient cells have a heightened sensitivity to DHODH inhibitors such as brequinar and leflunomide, that are already in clinical use. We confirm that DHODH inhibition selectively induces cell death in BCOR-mutant cells in multiple cellular models, in malignant and non-malignant cells, through chemical and genetic manipulation. Interestingly, we find that the dependency on DHODH does not stem from its role in de novo pyrimidine biosynthesis disruption. Rather, DHODH’s role in the electron transport chain, essential for mitigating reactive oxygen species, may be the physiological vulnerability that pushes BCOR-mutant cells toward cell death when DHODH is inhibited. DHODH inhibitors could be repurposed as targeted therapies for BCOR-mutant tumors, offering a promising strategy for precision medicine in AML and other cancers.

Doxorubicin (DOX) is an effective anticancer therapeutic but exhibits dose-dependent, potentially life-threatening cardiotoxicity. The specific mechanisms driving this cardiotoxicity are not fully understood but can include the induction of oxidative stress and subsequent cell death mechanism activation. This has prompted the exploration of NRF2, a master co-ordinator of antioxidant and largely cytoprotective pathways, as a potential approach for the alleviation of DOX-induced cardiotoxicity. Here, NRF2 was pharmacologically activated via CDDO-Me (hitherto referred to as CDDO) to reduce the negative consequences on AC16 human cardiomyocyte cell health and functions. NRF2 intracellular dynamics were quantitatively measured using live-cell imaging, demonstrating rapid (∼10 min) yet sustained (≥24 h) induction of NRF2 expression and functional downstream activity. Genetic perturbations of the NRF2-KEAP1 system highlight that CDDO acts specifically through NRF2 to exert AC16 cytoprotection from DOX whilst not promoting human lung and pancreatic cancer cell line viability. Via RNA-seq analysis, we reveal that CDDO dampens DOX-mediated effects on p53 signalling, apoptosis and ferroptosis. This study provides novel insight into NRF2 dynamics in the widely utilised AC16 cells whilst further elucidating the molecular mechanisms contributing to DOX cardiotoxicity and potential NRF2-orchestrated defence. Graphical abstract

Cerebral ischemic small vessel disease (SVD) is the leading cause of vascular dementia and a major contributor to stroke. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic form of familial SVD. CADASIL is caused by dominant missense mutations in Notch3, a receptor expressed in mural cells, including smooth muscle cells (SMCs) and pericytes. However, the cell-type specific contributions driving the CADASIL pathology remain unknown due to lack of animal models. Here, we generated two conditional knock-in mouse models carrying the CADASIL-causing Notch3 R170C mutation selectively in SMC and brain pericytes. Both Notch3 R 170 C models showed perivascular accumulation of the NOTCH3 extracellular domain, yet developed distinct neurovascular changes depending on the affected cell type. Pericyte-specific Notch3 R170C mice displayed pronounced region-selective microglial activation and vascular changes, whereas SMC-specific Notch3 R170C mice showed localized perivascular gliosis with minimal vascular remodeling. Proteomic profiling of isolated brain vessels revealed largely unique cell-specific responses. Pericytes Notch3 R170C expression dysregulated metabolic pathways, whereas SMC Notch3 R170C expression induced immune signaling related pathways. Integration with single-cell RNA-seq data revealed that many of the proteomic and phosphoproteomic shifts might also include brain endothelial cells, including metabolic changes in the presence of pericyte Notch3 R170C and inflammatory signaling in the presence of SMC-Notch3 R170C . Together, these findings define mural cell-specific mechanisms that contribute to the CADASIL-associated vascular pathology.

Modeling human epithelial diseases and developing cell-based therapies require robust methods to expand and manipulate epithelial stem and progenitor cells in vitro . Basal stem/progenitor cells from stratified epithelia can be expanded in 3T3-J2 fibroblast feeder cell co-culture systems, and the addition of the ROCK inhibitor Y-27632 enhances proliferation and culture longevity, a phenomenon described as ‘conditional reprogramming’. Here, we present a method incorporating the small molecule WS6 to further improve the proliferation and lifespan of cultured epithelial cells from multiple tissues, including airway, skin, and thymus. Cells maintained in this medium (‘EpMED’; FAD+Y+WS6) retain basal stem/progenitor cell identity and function, including the capacity to differentiate. We demonstrate their capacity to engraft in vivo in a tracheal transplantation model. In a second application, we generate clonal CRISPR-Cas9 genome edited nasal cultures, introducing targeted knockouts of DNAH5 or DNAI2 to create primary ciliary dyskinesia disease models. We anticipate that our method will have broad applications in epithelial cell biology, disease modeling, and regenerative medicine, while reducing reliance on immortalized or cancer cell lines and animal experimentation.