ABSTRACT Acute myeloid leukemia (AML) remains a major therapeutic challenge due to extensive disease heterogeneity and lack of cancer-specific antigens. ADGRE2 has emerged as a promising AML target with broad expression in AML patient blast and leukemic stem cell-enriched populations. However, comparable expression in healthy hematopoietic stem and progenitor cells (HSPCs) and myeloid lineages suggests a high susceptibility to on-target, off-tumor myelotoxicity with ADGRE2-targeted therapies. Guided by human genetics data identifying loss-of-function variants, we evaluated whether ADGRE2 is dispensable in hematopoietic stem cells as a protective approach for transplant-based shielding from ADGRE2-directed therapies. Using CRISPR-Cas9 and adenine base editors, we achieved high-efficiency ADGRE2 knockout (>94%) in HSPCs with corresponding protein loss without impairing cell viability, differentiation, and cytokine release in vitro , or long-term engraftment, multilineage differentiation, and persistence of gene editing in mouse xenografts. We also developed novel ADGRE2-specific chimeric antigen receptor (CAR) T cells that demonstrated potent cytotoxicity against AML cells, even at low antigen levels. Together, these findings establish ADGRE2 as a compelling AML target and provide a framework for hematopoietic stem cell transplant with protective gene editing to enable ADGRE2-directed immunotherapies while minimizing myelotoxicity.

ABSTRACT Heterotrimeric G proteins transduce signals from G protein coupled receptors, which mediate key aspects of neuronal development and function. Mutations in the GNAI1 gene, which encodes Gαi1, cause a disorder characterized by developmental delay, intellectual disability, hypotonia, and epilepsy. However, the mechanistic basis for this disorder remains unknown. Here, we show that GNAI1 is required for ciliogenesis in human cells and use C. elegans as a whole-organism model to determine the functional impact of seven GNAI1 -disorder patient variants. Using CRISPR-Cas9 editing in combination with robust cellular (cilia morphology) and behavioral (chemotaxis) assays, we find that T48I , K272R , A328P , and V334E orthologous variants impact both cilia assembly and function in AWC neurons, M88V and I321T have no impact on either phenotype, and D175V exerts neuron-specific effects on cilia-dependent sensory behaviors. Finally, we validate in human ciliated cell lines that D173V , K270R , and A326P GNAI1 variants disrupt ciliary localization of the encoded human Gαi1 proteins similarly to their corresponding orthologous substitutions in the C. elegans ODR-3 (D175V , K272R , and A328P ). Overall, our findings determine the in vivo effects of orthologous GNAI1 variants and contribute to mechanistic understanding of GNAI1 disorder pathogenesis as well as neuron-specific roles of ODR-3 in sensory biology. ARTICLE SUMMARY Gα subunits of heterotrimeric G proteins transduce signaling from G protein coupled receptors and play important roles in cell communication and complex behaviors. Mutations in the GNAI1 gene, which encodes Gαi1 protein, have been recently linked to a neurodevelopmental disorder; however, it remains unknown how GNAI1 patient mutations disrupt neuronal development or function to manifest in disease. We demonstrate that GNAI1 is required for ciliogenesis and use C. elegans as a whole-animal model in combination with human cells to identify cell-specific and conserved mechanisms of Gα dysfunction.

In C. elegans , the epidermis and its overlying extracellular matrix form a primary protective barrier, functioning as the first line of defense against environmental factors. To properly develop those cellular boundaries, a tightly controlled interaction of many molecules and pathways is needed. Mutant alleles of paqr-2 and iglr-2 (lipid homeostasis), dpy-21 (membrane trafficking), and sma-1 (actin-binding spectrin) result in hermaphrodite tail tip defects suggesting that this simple four-cell structure can serve as a sensitive model for the identification of pathways responsible for the establishment of cellular boundaries. With this in mind, we performed a small forward genetics screen of ∼800 ethyl methanesulfonate-mutagenized haploid genomes and identified 21 mutants with a tail end defective (Ted) phenotype. Whole genome sequencing of these mutants identified mutations in genes encoding either structural constituents of the cuticle itself (mostly collagen genes) or protein with regulatory functions. By using CRISPR/Cas9 we confirmed six novel alleles of ptr-18, paqr-2, nab-1, ncam-1, vab-9 and efn-4. We further characterized the loss of function allele ptr-18(et70) , which encodes a patch domain-containing (PTCHD) protein homologous to human PTCHD1. ptr-18(et70) has a significant effect on growth and development of the worms, while also increasing membrane permeability. Lipidomics analysis revealed no major alterations in membrane lipid composition, implicating cuticle defects as the primary cause of the observed permeability phenotype. Article summary We performed a forward genetics screen to identify hermaphrodite C. elegans mutants with a tail end defect with the goal to discover membrane and morphogenesis regulators. The screen of 800 haploid genomes revealed 21 tail end defective mutants, including 8 novel alleles of interesting regulator protein. We conclude that the tail tip phenotype can be useful in discovery of new pathways and interactions during development.

Uveal melanoma, the most common eye cancer in adults, remains limited to surgical intervention and chemotherapy, with a dismal survival rate that has not improved in over 50 years. To address this therapeutic impasse, we systematically analyzed public gene expression, RNAi, and CRISPR knockout datasets and identified RASGRP3 as an essential gene specifically for uveal melanoma. RasGRP3 is uniquely overexpressed and essential for survival in uveal melanoma cells, but dispensable in healthy cells. RasGRP3 remains “undruggable” due to its intracellular localization and lack of targetable binding pockets. To overcome this, we developed a CRISPR-Cas13d RNA-targeting therapeutic that specifically knocks down RasGRP3 mRNA. This Cas13d-based therapeutic mediates selective uveal melanoma killing through two synergistic mechanisms: (i) direct silencing of the essential RasGRP3 transcript, and (ii) collateral RNA degradation triggered by the cleavage of overexpressed RasGRP3. When delivered via optimized lipid nanoparticles encoding Cas13d mRNA and guide RNA, this strategy eliminated >97% of uveal melanoma cells while sparing healthy cells, including retinal pigment epithelial cells. This approach outperformed conventional Cas9 and siRNA methods in potency without inducing permanent genomic alterations. Our findings establish a RNA-targeting therapeutic for uveal melanoma and a framework for Cas13d-based interventions against broad “undruggable” cancers.

Abstract Background Chimeric antigen receptor (CAR)-based immunotherapies face significant translational challenges in solid tumor applications, particularly regarding manufacturing scalability, tumor targeting specificity, and antigen heterogeneity. This systematic review evaluates microbial systems as innovative platforms to address these limitations through synthetic biology-driven approaches, with a focus on bridging preclinical advances to clinical implementation. Results Analysis of 389 peer-reviewed studies (2015–2025) reveals that engineered probiotic strains (e.g., Escherichia coli Nissle 1917) achieve selective tumor colonization while functioning as programmable factories for: 1. Synthetic antigen production and single-chain variable fragment (scFv) expression, 2. Costimulatory domain delivery enabling antigen-agnostic CAR-T activation, 3. Tumor microenvironment modulation via immunostimulatory chemokines. Microbial platforms demonstrate superior manufacturing economics (70–90% cost reduction vs. conventional methods) and enhance CAR-T functionality through epigenetic reprogramming by microbial metabolites (e.g., short-chain fatty acids). CRISPR/Cas-engineered genetic circuits further enable precise spatiotemporal control of therapeutic payloads. Conclusions Microbial systems represent transformative platforms for scalable, programmable CAR immunotherapy with significant potential for solid tumor targeting. Key barriers to clinical translation include biocontainment challenges, incomplete mechanistic understanding of tumor homing specificity, and safety validation requirements. Strategic integration of synthetic biology with microbial chassis offers a viable pathway toward accessible next-generation cancer therapies.

Abstract Background Toxoplasmosis, caused by Toxoplasma gondii ( T. gondii ), poses a significant global health threat with no commercial vaccine available. The parasite's egress process, which bridges its intracellular replication cycles and is critical for survival and dissemination, is tightly regulated by calcium. Notably, the T. gondii EF-hand domain-containing protein (Efhc) exhibits the highest Ca²⁺ binding affinity among its calcium-binding proteins. Methods CRISPR/Cas9 was used to generate a conditional knockout strain (TgEfhc3-C-AID). Phenotypic assays (plaque, intracellular proliferation, egress, invasion and murine virulence) were used to assess its impact on tachyzoites growth and development. Subsequently, TgEfhc3 antigenicity was analyzed using DNAstar software, Immunofluorescence assays and Western blots. Recombinant TgEfhc3 (rTgEfhc3) proteins, expressed in E. coli , was subcutaneously administrated to BALB/c mice to evaluate its protective efficacy against acute toxoplasmosis. Immune mechanisms induced by rTgEfhc3 were analyzed by measuring: serum IgG/IgG subclasses (IgG1, IgG2a) and splenic T cell cytokines (IL-4, IFN-γ, IL-10) by ELISA; the frequencies of CD4 + and CD8 + T cells by flow cytometry. Results Genetic deletion severely impaired tachyzoite proliferation, egress, and invasion, indicating its essentiality in T. gondii biology. Furthermore, recombinant TgEfhc3 was evaluated as a subunit vaccine in mice model, and was subsequently shown to protect against acute T. gondii infection. Immunization induced high levels of anti- T. gondii IgG and subclasses, enhanced Th1/Th2 cytokine production (IL-4, IFN-γ, IL-10) in splenic T lymphocytes, and stimulated robust CD4⁺ T cell proliferation. This elicited complex cellular and humoral immunity significantly prolonged survival time following acute T. gondii infection. Conclusions This study identifies that TgEfhc3 is crucial for T. gondii tachyzoite growth and development, providing new insights into infection mechanisms. Given the partial protective immunity conferred, TgEfhc3 warrants consideration as a component in future toxoplasmosis vaccine strategies.

Genome structural variants (SVs) comprise a sizable portion of functionally important genetic variation in all organisms; yet, many SVs evade discovery using short reads. While long-read sequencing can find the hidden SVs, the role of SVs in variation in organismal traits remains largely unclear. To address this gap, we investigate the molecular basis of 50 classical phenotypes in 11 Drosophila melanogaster strains using highly contiguous de novo genome assemblies generated with Oxford Nanopore long reads. These assemblies enabled the creation of a pangenome graph containing comprehensive, nucleotide-resolution maps of SVs, including complex rearrangements such as the interchromosomal inverted duplication Dp(2;4)eyD and large tandem duplications at the Bar locus. We uncovered new candidate causal mutations for 15 phenotypes and new molecular alleles for 2 mutations comprising tandem duplications, transposable element (TE) insertions, and indels. For example, we mapped the tarsal joint defect Ablp eyD to an 8 kb Roo retrotransposon insertion into an intergenic enhancer, a finding validated via CRISPR-Cas9. The wing vein phenotype plexus (px 1 ) was linked to a 1.5 kb partial tandem gene duplication, and the century-old Curved (c 1 ) wing phenotype was linked to a 7.5 kb DM412 retrotransposon inserted into the coding sequence of the muscle protein gene Strn-Mlck . We also unveiled 8 SV alleles of previously identified causal genes, including previously uncharacterized SVs underlying the extensively studied white and yellow phenotypes. Overall, 67.4% of the genes causing phenotypic changes harbored candidate SVs over 100 bp, whereas only 28% is expected based on euchromatic SVs. Our data, based on the 50 Drosophila phenotypes, 44 of which are strongly deleterious, suggests a disproportionately larger contribution of SVs to deleterious changes in visible phenotypes in Drosophila .

Neutrophils, the first cells to arrive at the site of inflammation, are rather short-lived cells and thus have to be constantly replenished. During neutrophil development, vesicle dynamics need to be fine-tuned and impaired vesicle trafficking has been linked to failure in neutrophil maturation. Here, we characterized the role of VPS18 as a central core component of CORVET & HOPS tethering complexes for neutrophil development. Using CRISPR/Cas9-engineered Hoxb8 cells with heterozygous mutations in Vps18 , we found that VPS18 deficiency interfered with neutrophil development due to tethering complex instability. As a result, vesicle dynamics were impaired with a strong increase in LC3-II and p62 levels, indicating autophagosome accumulation and reduced autophagic flux. With transmission electron microscopy, we verified the increase in autophagosomes and also found irregularly shaped vesicular structures in Vps18 mutants. Subsequently, Vps18 mutant neutrophil progenitors underwent premature apoptosis. We described a novel patient with a heterozygous stop-gain mutation in VPS18 suffering from neutropenia and recurrent infections. To verify our findings in the human system, we used human induced pluripotent stem cells (iPSCs). Upon differentiation into neutrophils, loss of VPS18 resulted in an almost complete absence of iPSC-derived developing neutrophils. Heterozygous VPS18 mutant and patient mutation-harboring iPSCs were characterized by strongly reduced numbers of developing neutrophils. Zebrafish larvae with heterozygous mutations in vps18 were also characterized by significantly reduced neutrophil numbers. This study shows the pivotal impact of VPS18 for adequate vesicle dynamics during neutrophil development which might be relevant in the context of vesicle trafficking during granulopoiesis and congenital neutropenia.

Abstract Hereditary persistence of fetal hemoglobin (HPFH) represents a clinically proven therapeutic model for β-hemoglobinopathies, where in persistent γ-globin (HBG1/HBG2) production compensates for impaired β-globin synthesis. To capitalize on this naturally occurring mechanism, we engineered a precise Clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) strategy targeting HBG regulatory elements to therapeutically reactivate fetal hemoglobin. Through optimized Cas9: sgRNA ribonucleoprotein (RNP) delivery, we implemented a dual-editing approach: (1) B-cell lymphoma/leukemia 11A (BCL11A) binding site disruption to alleviate transcriptional repression and (2) HPFH-associated deletion recapitulation. This strategy achieved robust fetal hemoglobin (HbF) reactivation in multiple experimental systems, including K562 erythroleukemia cells and primary hematopoietic stem/progenitor cell (HSPC)-derived erythroid cultures. The successful translation of this approach could provide a transformative treatment paradigm for β-thalassemia and sickle cell disease patients.

Abstract Many common diseases have a polygenic architecture. The responsible alleles are thought to mediate risk by disturbing gene regulation in most cases, however, the precise mechanisms have been elucidated only for a few. Here, we investigated the 16q23.3 genomic locus, which genome-wide significantly associates with coronary artery disease, a globally leading cause of death caused by accumulation of lipid-rich inflammatory plaques in the arterial wall. The locus harbors CDH13, whose mRNA and protein we found to be suppressed in atherosclerotic human and mouse arteries. Loss-of-function(LoF) variants of CDH13 were associated with detrimental cardiovascular phenotypes in the UK Biobank. Its knock-out increased plaque-sizes in Cdh13 -/- / Apoe -/- mice compared to Apoe -/- mice on a Western diet. After establishing an atheroprotective role of CDH13 , we studied its regulation. Integration of population genomic and transcriptomic datasets by GWAS-eQTL colocalization analysis identified CDH13 and four long non-coding RNAs (lncRNAs) as candidate causal genes at the 16q23.3 locus. dCas13-mediated RNA immunoprecipitation revealed that the lncRNA CDH13-AS2 binds to CDH13 mRNA in human endothelial cells (ECs). Its CRISPR/Cas9-based knockout in ECs was atherogenic, whereas dCas9-based transcriptional activation (CRISPRa) of CDH13-AS2 was atheroprotective; effects that were found to be mediated by the stability of CDH13 mRNA. To further understand how the CDH13-AS2 protects the mRNA we searched in silico and screened in vitro for microRNAs (miRNAs) that bind to CDH13 3’UTR. Indeed, four miRNAs, miR-19b-3p, miR-125b-2-3p, miR-433-3p, and miR-7b-5p, were found experimentally to accelerate CDH13 mRNA degradation, an effect that was neutralized by CRISPRa of CDH13-AS2 . Taken together, our study demonstrates an interplay of miRNAs, lncRNAs, and mRNA, which modulates the abundance of an atheroprotective protein in endothelial cells, which may offer a new therapeutic target for coronary artery disease.

Abstract Climate change is becoming a great challenge to sustainable crop production in sub-Saharan Africa. Conventional control methods are becoming ineffective against high-risk pests such as Spodoptera frugiperda , Bactrocera dorsalis , and Maruca vitrata . To overcome such challenges, we created an AI-CRISPR approach that combines real-time insect monitoring, climate-adaptive gene targeting, and field-tested suppression measures.The study utilised climatic modeling, host-pest phenology mapping, and population genomics to identify stable gene targets in life stages and climatic stress conditions. CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9) constructs were developed to break the fundamental gene controlling reproduction (vitellogenin), host detection (odorant-binding proteins), and thermotolerance, enhancing detection of pests by 88 percent and decreasing the use of pesticides by 35 percent.Simulated climate stress knockouts on specific traits mitigated oviposition of S. frugiperda by 72%, B. dorsalis fruit infestation by 65% and M. vitrata larval mortality by 81%. In Abuja and Makurdi, field trials demonstrated that population reduction of pests was at 83.4, 75.1, and 697.7 percent, respectively, and this resulted in an increase in the yield of crops by 28 percent and also a reduction of 40 percent in post-harvest losses. AI models forecast that the resurgence of the pest will occur by 61 percent after 2040–2050.The research constructed an AI-CRISPR-IPM pipeline, which can bolster climate resilience, diminish chemical reliance, and fit into local agriculture frameworks, providing an environmentally viable answer to African food security in a world that is warming.

SUMMARY Extracellular alkalinization has long been recognized as a hallmark of plant cell-surface receptor activation, including during pattern-triggered immunity (PTI); yet the mechanisms driving elicitor-induced alkalinization and its role in immune signaling remain unclear. Here, we demonstrate that inhibition of autoinhibited H + -ATPases (AHAs) is required for elicitor-induced extracellular alkalinization. This alkalinization is essential for immune signaling mediated by diverse plasma membrane-localized receptor kinases (RKs) through modulation of ligand-receptor interactions. Notably, RKs transduce elicitor-triggered signaling via BOTRYTIS-INDUCED KINASE 1 (BIK1), which inhibits AHA activity by disrupting AHA-GENERAL REGULATORY FACTOR (GRF) interactions through a conserved phosphorylation event. Interestingly, this pathway is crucial for cell wall damage (CWD) responses involving the RK MALE DISCOVERER 1-INTERACTING RECEPTOR LIKE KINASE 2 (MIK2) and its ligand, SERINE RICH ENDOGENOUS PEPTIDE 18 (SCOOP18). Our findings reveal a conserved phospho-regulatory pathway that governs extracellular alkalinization to coordinate plant immune signaling, offering new insights into plant stress resilience.

ABSTRACT Carbon and zinc (Zn) metabolism are intrinsically connected in phototrophs, as crucial components involved in CO 2 assimilation, like carbonic anhydrases, are highly abundant Zn proteins. Utilizing these and other proteins, the eukaryotic green algae Chlamydomonas reinhardtii can maintain phototrophic growth in low CO 2 environments by inducing a carbon concentrating mechanism (CCM). In this work we show that Chlamydomonas dynamically increases its Zn content to accommodate the higher intracellular Zn demand in low CO 2 environments. This increase requires the presence of Cia5, a major regulator of the CCM in Chlamydomonas. How Cia5 regulates expression of thousands of low CO 2 -inducible genes remains enigmatic, its transcript and protein abundance is unchanged in different CO 2 environments, even in the presence of an additional reduced carbon source, acetate. We show here that the Cia5 protein is not present in Zn-limitation, despite CIA5 transcription being unchanged. We used a CRISPR knock-in approach to express Cia5-HA from its endogenous locus and used two independent Cia5-HA expressing strains for affinity purification and identified a protein belonging to a conserved family of metal binding GTPases, ZNG3, as a constitutive interaction partner. Like Cia5, ZNG3 is constitutively expressed, co-expressed with Cia5 along the diurnal cycle and is Cia5-dependently induced in low CO 2 environments. Surprisingly, zng3 mutants do not phenocopy cia5 mutants and grow well in low CO 2 conditions. Instead, zng3 mutants are unable to grow like wildtype if excess carbon is available in the form of high CO 2 or acetate. Transcriptomics of wildtype and zng3 mutants grown with different carbon sources revealed that transcriptional induction of the majority of genes involved in the CCM is maintained in low CO 2 grown zng3 mutants, while the degree of induction in a subset of LCI genes is reduced ( HLA3 , CAH4 and CAH5 ). Genes encoding proteins involved in plastid quality control were induced in zng3 mutants grown on acetate and high CO 2 , as well as other, related metallochaperones. We hypothesize that Zn trafficking towards the plastid is mis regulated in zng3 mutants resulting in protein mis-metalation and unfolding. Taken together, we propose that ZNG3 and Cia5 coordinate Zn and CO 2 metabolism, affecting intracellular Zn trafficking and modulate the CO 2 response.

ABSTRACT Typically diagnosed late, when systemic metastasis has already occurred, pancreatic ductal adenocarcinoma (PDAC) has one of the worst 5-year survival rates of any cancer type. For many patients with advanced disease, current chemotherapy regimens offer only modest benefit despite significant toxicity and surgical resection, the only treatment option with curative potential, is not possible. Therefore, while new treatments are much needed, diagnosing patients at an ‘earlier’ disease stage when surgery remains possible and the window of opportunity for treatment response is longer will be critical to improving patient outcomes. In this regard, the identification of biomarkers from biospecimens that can be easily sampled from patients remains the focus of considerable research, however success has not been forthcoming. Using a suite of novel genetically defined murine isogenic models of early PDAC, engineered using CRISPR-Cas9 gene editing, we sought to address whether loss-of-function mutations in common driver genes, and thus the genetic heterogeneity inherent to the disease, may represent an important confounding factor in the identification of a one-size-fits-all biomarker suitable for early detection. Focussing on the multi-omics analysis of blood, we show that both loss of Cdkn2a and / or Smad4 on the background of a Kras G12D Trp53 -/ - genotype has profound effects on the profile of differentially expressed RNA species including protein coding RNAs, lncRNAs, snoRNAs, scRNAs, snRNAs and miRNAs, and on plasma protein expression, when compared to both healthy controls and chemically induced pancreatitis. In addition, we find that loss of Smad4 , a genomic event that occurs following progression from PanIN to PDAC, substantially limits the availability of blood biomarkers. These findings identify the need to move towards genotype-specific biomarker signatures and uncover a potential role for Smad4 loss in limiting opportunities for the early detection of pancreatic cancer.

Since 2021, subclade 2.3.4.4b A(H5N1) high pathogenicity avian influenza (HPAI) viruses have undergone changes in ecology and epidemiology, causing a panzootic of unprecedented scale in wild and domestic birds with spill-over infections and perceptible transmission in a range of mammalian species, raising concern over zoonotic potential. HPAI viruses readily exchange gene segments with low pathogenicity avian influenza viruses via reassortment, a mechanism that facilitates pronounced phenotypic change. Observations suggest changes in the seasonality and host range of panzootic viruses, however, data on the role of reassortment in determining such features are limited. Using phylodynamic approaches, we describe the emergence of the panzootic lineage and using a novel global genotype classification system we describe the subsequent emergence and global structuring of genotypes generated by reassortment. Focusing on evolutionary dynamics in Europe, we show reassortment has produced high fitness genotypes with enhanced capacity for transmission and further we show such advantages can be host-dependent, contrasting successful generalist genotypes with a specialist lineage (EA-2022-BB) adapted to birds of the order Charadriiformes. Experimental investigation of NS1-mediated shutoff indicates this Charadriiformes-specialist does not inhibit host cellular gene expression and hamper the defences of more typical hosts such as water- and land-fowl. We attribute this primarily to variation at position 127 of the NS1 protein. Our results emphasise that reassortment has driven phenotypic change, affected viral fitness, and caused diversification of host specificity and seasonality. Such factors should be considered in studies that seek to identify drivers of HPAI spread and map spillover risk. Additionally, relaxation of host specialisation, ecological diversification, and potential endemicity in atypical host populations present new reassortment opportunities that could result in further novel phenotypes.

Abstract Pooled CRISPR-Cas9 screens have emerged as powerful functional genomic tools. However, they typically rely on aggregate-level or transcriptomic outputs, which limits their ability to capture spatially resolved phenotypes. We developed spatial phenotype analysis of CRISPR-Cas9 screens (spaCR), an open-source software package that integrates deep-learning single-cell image classification with barcode-based, well-level genotype mapping to link pooled genetic perturbations to spatial phenotypes. Unlike optical pooled screening or in situ genotyping methods, spaCR requires only standard imaging and sequencing platforms, eliminating the need for specialized equipment. To guide experimental design, we developed spaCRPower, a statistical simulation tool that models screen parameters and estimates power for detecting genotype–phenotype associations. As a proof-of-concept, we applied spaCR to a pooled Toxoplasma mutant library to identify parasite effectors involved in hijacking the host ESCRT machinery. We identified both an established and a previously uncharacterized genetic determinant, EAF1, highlighting spaCR as a powerful tool for mapping genotype–phenotype relationships.

Abstract Autoimmune diseases represent a class of complex, multifactorial disorders characterized by the immune system’s aberrant recognition of self-antigens as foreign, leading to chronic inflammation, tissue damage, and progressive organ dysfunction. Despite advances in immunosuppressive pharmacology, current therapies remain largely non-specific, systemically cytotoxic, and incapable of achieving long-term immune tolerance. In response to this therapeutic gap, we present SynAuto-X—a synthetic biology platform integrating a programmable CRISPR-dCas9-based gene circuit designed for the conditional silencing of pro-inflammatory genes that drive autoimmune pathology. SynAuto-X employs a modular genetic architecture based on catalytically inactive Cas9 (dCas9) fused to the KRAB (Krüppel-associated box) repressor domain. This fusion enables precise transcriptional repression of key cytokine and co-stimulatory genes (e.g., IL17A, IFNG, TNFA, CD80) through CRISPR interference (CRISPRi), directed by customizable single-guide RNAs (sgRNAs). Gene silencing is driven by inflammationresponsive promoters, such as NF-B or STAT1 elements, allowing context-specific activation of the repression cascade only in immune cells undergoing aberrant activation. The architecture supports single-input singleoutput (SISO) and combinatorial logic designs, enabling multigene targeting, threshold-dependent activation, and Boolean gating of inflammatory response elements. A key innovation within SynAuto-X is the integration of a drug-inducible kill switch for enhanced biosafety. Utilizing a chemically controlled inducible Caspase-9 (iCasp9) domain responsive to the synthetic small molecule AP1903, the system enables externally triggered apoptosis of transduced cells upon detection of adverse immune modulation or unintended off-target effects. The dynamics of this safety module are modeled through coupled differential equations, representing CID-mediated dimerization and apoptotic cascade activation, with tunable parameters for dose-responsive control. Additionally, the full circuit is encoded within a lentiviral vector framework, optimized via Benchling and CHOPCHOP for minimal off-target sgRNA activity, promoter leakiness, and vector immunogenicity. SynAuto-X represents a programmable, safe, and cell-specific alternative to broad-spectrum immunosuppression. By combining precision CRISPR interference, logic-based gene circuit design, and fail-safe therapeutic control, SynAuto-X advances the frontiers of autoimmune treatment toward intelligent, synthetic immune regulation. This work lays the foundation for a new class of gene circuit-based therapeutics capable of rewiring immune responses with the specificity of molecular computation and the safety of on-demand self-destruction.

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates apoptosis, autophagy, inflammation, and intracellular pathogen survival. While S1P signaling has been implicated in Leishmania donovani infection, the specific roles of its biosynthetic enzymes Sphingosine kinases SphK1 and SphK2 in host macrophage remains poorly defined. Here, we delineate the role of SphK1 in modulating host-pathogen interactions using CRISPR/Cas9-mediated knockout and pharmacological inhibition in THP-1 macrophages. We generated and validated CRISPR constructs targeting SphK1 (promoter, SBS, exon 6) and SphK2 (exon 3). SphK1 Knockout was confirmed at transcript and protein levels, accompanied by a marked reduction in SphK1 enzymatic activity and S1P levels. Functionally, SphK1 knockout macrophages exhibited decreased intracellular L. donovani burden, elevated TNF-α, and reduced IL-10, and increased autophagic and apoptotic markers, suggesting a pro-inflammatory, cell-death-prone state. Pharmacological inhibition using the selective SphK1 inhibitor, PF-543 recapitulated these findings, showing reduced phosphorylated SphK1, enhanced p38MAPK activation, and augmented autophagy and apoptosis. Conversely, the SphK2 inhibitor ABC294640 had minimal effect, reinforcing the predominant role of SphK1. Together, our study identifies SphK1 as a critical host factor that facilitates L. donovani survival by modulating lysosomal stress, immune evasion, and cell fate pathways. Targeting SphK1-S1P signalling may offer a novel therapeutic approach for visceral leishmaniasis.

Mounting evidence implicates microglia in neurodegeneration, but linking disease-associated genetic variants to target genes and cellular phenotypes is hindered by the inaccessibility of these cells. We differentiated 261 human iPSC lines into microglia-like cells (iMGL) in pools with phenotypic (differentiation, phagocytosis and migration) and single-cell transcriptomic readouts. Burden analysis of deleterious variants detected 36 genes influencing microglial phenotypes. Expression quantitative trait locus (eQTL) analysis found 7,121 eGenes, and 79 colocalizations across four neurodegenerative disease GWAS, half of which had limited prior evidence of causality. Integration of eQTL and phenotypic associations highlighted the role of disease-relevant variants including LRRK2 and TREM2 acting via microglial phagocytosis. A coupled CRISPR screen identified a role of TREM2 in phagocytosis and highlighted the importance of cellular state in directionality of phenotype. By contextualizing variant effects within disease-relevant microglial states, we provide a comprehensive framework for interpreting the function of risk loci in neurodegenerative disorders.

ABSTRACT The emergence of SARS-CoV-2 has posed significant threats to global health, particularly for the older population. Similarly, common human coronaviruses, such as HCoV-229E, which typically cause mild cold-like symptoms, have also been linked to severe diseases, underscoring the need to understand virus-host interactions and identify host factors contributing to viral pathogenesis and disease progression. In this study, we performed a genome-wide CRISPR knockout screen using HCoV-229E and identified Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) as a potent restriction factor. Mechanistically, UHRF1 suppressed HCoV-229E infection by downregulating the expression of its cell entry receptor, aminopeptidase N (APN), through promoter hypermethylation. Focused CRISPR activation screens of UHRF1-downregulated genes confirmed the critical role of APN in HCoV-229E infection and identified additional genes (e.g., SIGLEC1, PLAC8, and heparan sulfate biosynthesis genes) contributing to the restrictive functions of UHRF1. Transcriptomic and single-cell RNA sequencing analysis revealed that UHRF1 expression decreases with age, negatively correlating with increased APN expression. This age-related decline in UHRF1 was further validated in primary alveolar macrophages isolated from elderly individuals, which exhibited heightened susceptibility to HCoV-229E infection compared to those from younger individuals. Our findings highlight UHRF1 as a key age-related host defense factor against coronavirus infection and provide novel insights into the epigenetic regulation of viral entry receptors.

Sporozoites of Plasmodium falciparum , the deadliest malaria parasite, are transmitted into the skin by infected mosquitoes and migrate to the liver to initiate infection. There, they invade hepatocytes and develop into exoerythrocytic merozoites that, eventually, enter the bloodstream and invade erythrocytes, leading to malaria. The parasite journey involves cell traversal, where sporozoites transiently enter and exit host cells beginning in the skin, lysing membranes to move deeper into tissue and evade immune cell destruction. After reaching the liver and traversing several hepatocytes, sporozoites productively invade a final hepatocyte to establish liver-stage infection. The molecular mechanisms underlying traversal, invasion, and intracellular development remain incompletely understood, particularly with respect to host determinants. To address this, we engineered human HC-04 hepatocytes, the only known cell line supporting P. falciparum liver-stage development, to express Cas9-mCherry, enabling CRISPR-based functional genomics studies. We validated Cas9 activity and demonstrated successful guide-RNA-directed gene disruption via non-homologous end joining in HC-04 Cas9+ (clone 2B3) cells. Optimized traversal and invasion assays with HC-04 2B3 cells led to a robust cytometric assay suitable for screening human genes involved in P. falciparum infection. As proof-of-concept, we performed a small screen involving disruption of 10 human genes previously implicated in infection by bacterial and viral pathogens, confirming utility of this platform. While no new host factors were identified for malaria parasites in this initial study, we have developed a tractable system for genome-wide CRISPR screens to uncover novel hepatocyte biology and host determinants of infection by liver-tropic pathogens.

SUMMARY EN - Goldenberry ( Physalis peruviana ) produces sweet, nutritionally-rich berries, yet like many minor crops, is cultivated in limited geographical regions and has not been a focus of breeding programs for trait enhancement. Leveraging knowledge of plant architecture-related traits from related species, we used CRISPR/Cas9-mediated gene editing to generate a compact ideotype to advance future breeding efforts and agricultural production. Goldenberry growers will benefit from these compact versions because it optimizes per plot yield, facilitating larger-scale production to meet rising consumer popularity and demand. SP - La uchuva ( Physalis peruviana ) produce frutos dulces y ricos en nutrientes, pero, igual que muchos cultivos minoritarios, se cultiva en zonas geográficas limitadas y no ha sufrido un proceso de mejoram. Aprovechando conocimientos sobre rasgos relacionados con la arquitectura vegetal de especies relacionadas, hemos usado edición génica mediante CRISPR/Cas9 para generar un ideotipo compacto para promover futuros esfuerzos en su mejora y en producción agrícola. Los productores de uchuva se podrán beneficiar de estas versiones compactas ya que optimiza el rendimiento por parcela, facilitando así la producción a una mayor escala para cubrir la creciente popularidad y demanda de los consumidores.

Abstract Insulin-like growth factor 1 (IGF-1) is known to promote cancer cell proliferation, but its role in metastasis remains incompletely understood. Autophagy, a key regulator of cancer cell behavior, plays a significant role in colorectal cancer (CRC) progression. Our previous transcriptomic analysis identified autophagy-related genes and insulin-like growth factor 1 receptor (IGF-1R) among the most differentially expressed in advanced versus early-stage CRC. However, the mechanistic contribution of IGF-1R to autophagy-driven CRC metastasis has not been fully elucidated. In this study, we investigated the functional interaction between IGF-1R signaling and autophagy in CRC progression using a panel of CRC cell lines, including HCT116 cells with CRISPR/Cas9-mediated knockout of ATG5 and ATG7. Our results demonstrate that IGF-1 stimulation enhances autophagic flux, whereas IGF-1R knockdown suppressed autophagic activity. Notably, dual inhibition of IGF-1R and autophagy led to a marked reduction in CRC cell migration and invasion. In ATG5-/- and ATG7-/- cells, IGF-1R silencing significantly downregulated mesenchymal markers Vimentin, Slug, and Snail while upregulating the epithelial marker E-cadherin. Additionally, combined inhibition increased size and number of focal adhesion molecules, such as paxillin and zyxin. These findings highlight the synergistic effect of IGF-1R and autophagy inhibition in suppressing EMT and metastatic potential in CRC cells, suggesting that this combinatorial approach may represent a promising therapeutic strategy for metastatic CRC. Further studies are warranted to delineate the underlying molecular mechanisms and evaluate these findings' translational potential in clinical settings.

Abstract Gaucher disease type 1 is a lysosomal storage disorder caused by GBA1 mutations that reduce glucocerebrosidase activity, leading to glycolipid buildup, particularly in macrophages. To develop a curative approach, we established a high-efficiency genome editing platform for human and murine hematopoietic stem-progenitor cells using CRISPR/Cas9, recombinant adeno-associated virus serotype 6. To enhance homology-directed DNA repair while minimizing genotoxicity, we incorporated a new 53BP1 inhibitor, a ubiquitin variant that promotes DNA end resection and significantly increases editing efficiency. This enabled precise insertion of a human GBA1 transgene—driven by a macrophage-specific promoter—into the mouse Rosa26 and human CCR5 safe-harbor loci. To assess efficacy, we established a rapidly progressive Gaucher disease mouse model by inducing hematopoietic-specific Gba1 deletion in a D427V background. Transplantation of edited cells corrected hematologic and visceral abnormalities, normalized lipid storage, and was effective under myeloablative and reduced-intensity busulfan conditioning. Notably, therapeutic benefit was achieved with only ~ 3% edited allele engraftment. These findings offer strong proof-of-concept for ex vivo genome editing as a mutation-agnostic, potentially curative strategy for Gaucher disease and support its clinical advancement.

Autophagy degrades cellular material by sequestering it in autophagosomes, which form de novo from precursors called phagophores. Phagophore assembly and expansion require ATG9A-positive seed compartments, the lipid transfer protein ATG2A, and the class III phosphatidylinositol 3-phosphate kinase complex I (PI3KC3-C1). PI3KC3-C1 synthesizes phosphatidylinositol 3-phosphate (PI3P), a key lipid that drives downstream processes for phagophore expansion, including ATG8 lipidation. We find that ATG9A compartments contain only traces of phosphatidylinositol (PI), likely insufficient for efficient PI3P production or recruitment of PI3P-binding effectors. Nevertheless, ATG2A is recruited to these compartments and mediates lipid transfer, including PI, into them. Remarkably, even without detectable PI3P, ATG9A compartments can support ATG8 lipidation, and ATG8 proteins themselves enhance ATG2A-mediated lipid transfer. In cells, ATG2A is essential for the appearance of PI3P on ATG9A compartments. Together, these findings support a model in which a lipid transfer-driven feedback loop, rather than pre-existing PI content, is required to activate ATG9A compartments for phagophore expansion. Teaser A feedback loop driven by lipid transfer activates ATG9A compartments for autophagosome biogenesis.