ABSTRACT The rapid evolution of novel functions requires targeted mutagenesis to avoid harmful mutations. Diversity-generating retroelements (DGRs) are natural systems that accelerate the evolution of diverse bacterial functions through targeted hypermutation. Here, we establish a method utilizing DGRs coupled to recombineering (DGRec), enabling the diversification of any sequence of interest in E. coli . DGRec can programmably diversify specific residues by leveraging the high error rate of the DGR reverse-transcriptase at adenines. We perform a detailed characterization of the reverse-transcriptase biases, highlighting how it maximizes the exploration of the sequence space while avoiding nonsense mutations. Applied to the phage λ GpJ receptor binding domain, and to its lamB receptor, DGRec created diverse variants enabling E. coli to evade infection, and λ to reinfect lamB mutants.

We describe a scalable and cost-effective sgRNA synthesis workflow that reduces costs by over 70% through the use of large pools of microarray-derived oligos encoding unique sgRNA spacers. These sub-pool oligos are assembled into full-length dsDNA templates via Golden Gate Assembly before in vitro transcription with T7 RNA polymerase. RNA-seq analysis reveals severe biases in spacer representation, with some spacers being highly overrepresented while others are completely absent. Consistent with previous studies, we identify guanine-rich sequences within the first four nucleotides of the spacer, immediately downstream of the T7 promoter, as the primary driver of this bias. To address this issue, we introduced a guanine tetramer upstream of all spacers, which reduced bias by an average of 19% in sgRNA libraries containing 389 spacers. However, this modification also increased the presence of high-molecular-weight RNA species after transcription. We also tested two alternative bias-reduction strategies: compartmentalizing spacers within emulsions and optimizing DNA input and reaction volumes. Both methods independently reduced bias in 2,626-plex sgRNA libraries, though to a lesser extent than the guanine tetramer approach. These advancements enhance both the affordability and uniformity of sgRNA libraries, with broad implications for improving CRISPR-Cas9 screens and optimizing guide RNA design for other CRISPR and nuclease systems.

Abstract Phosphorus (P) is a crucial macronutrient and its deficiency severely limits plant growth and yield. Although multiple inorganic phosphate (Pi) signaling regulators have been identified, the function of them in plant development and flowering time regulatory remains inadequately characterized in C4 model species like Setaria italica . Here, CRISPR/Cas9-generated SiPHO2 knockout lines exhibited disrupted Pi homeostasis, and the lines showed shoot Pi accumulation, leaf tip necrosis, modified root architecture and reduced yield compared with wildtype (Ci846) under Pi deficient conditions. Transcriptome analysis suggested these phenotypic abnormalities might due to expression patterns alteration of Pi starvation-responsive genes. Notably, SiPHO2 knockout lines displayed earlier heading date under Pi deficiency but delayed heading date under normal conditions compared to Ci846 plants. Expression profiling and transgenic functional verification revealed that the heading date reversal correlated with the expression pattern of FLOWERING LOCUS T c ( SiFTc ), rather than SiFTa , which is the closest homolog of Heading date 3a ( OsHd3a ). This study identifies a novel flowering regulator as a potential target for coordinating phosphorus-mediated heading date regulation and yield production. Our findings elucidate genetic mechanisms underlying phosphorus-dependent developmental regulation and propose a strategic approach for improving crop yield under Pi starvation.

ABSTRACT Long non-coding RNAs (lncRNAs) can regulate gene expression. Some are essential for organismal development and physiology and can contribute to diseases including cancer. Whilst most lncRNAs exhibit little sequence similarity, conservation of lncRNA transcription relative to neighbouring protein-coding genes suggests potential functional significance. Most positionally equivalent lncRNAs are uncharacterized and it remains unclear whether they exert similar roles in distant species. Here, we identified syntenic melanoma-associated lncRNAs predicted to be components of the MITF gene regulatory network in human melanoma, with positionally equivalent transcripts in zebrafish. We prioritized Differentiation Antagonizing Non-Protein Coding RNA ( DANCR ), a cancer-associated lncRNA critical for maintaining somatic progenitor cells in human models, for functional investigation. Dancr is a multi-exonic, cytoplasmically-enriched lncRNA transcribed from syntenic regions in the human and zebrafish genomes. MITF and c-MYC, key melanoma transcription factors, regulate human DANCR expression and melanoma patients with high DANCR display significantly decreased survival. DANCR is a melanoma oncogene that controls cancer-associated gene expression networks and promotes human melanoma cell proliferation and migration. Zebrafish dancr is dynamically expressed across multiple different cell types in the developing embryo, regulates genes involved in cell death, and is essential for embryonic development. Our work suggests that cancer-critical lncRNAs such as DANCR , expressed from similar regions in vertebrate genomes, may regulate related genes and processes involved in both embryonic development and tumorigenesis across species.

Pooled processing, in which cells from multiple sources are cultured or captured together, is an increasingly popular strategy for droplet-based single cell sequencing studies. This design allows efficient scaling of experiments, isolation of cell-intrinsic differences, and mitigation of batch effects. We present CellBouncer, a computational toolkit for demultiplexing and analyzing single-cell sequencing data from pooled experiments. We demonstrate that CellBouncer can separate and quantify multi-species and multi-individual cell mixtures, identify unknown mitochondrial haplotypes in cells, assign treatments from lipid-conjugated barcodes or CRISPR sgRNAs, and infer pool composition, outperforming existing methods. We also introduce methods to quantify ambient RNA contamination per cell, infer individual donors’ contributions to the ambient RNA pool, and determine a consensus doublet rate harmonized across data types. Applying these tools to tetraploid composite cells, we identify a competitive advantage of human over chimpanzee mitochondria across 10 cell fusion lines and provide evidence for inter-mitochondrial incompatibility and mito-nuclear incompatibility between species.

Cystic fibrosis (CF) is caused by homozygous mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene, resulting in multi-organ dysfunction and decreased lifespan and quality of life. A durable cure for CF will likely require a gene therapy approach to correct CFTR. Rapid advancements in genome editing technologies such as CRISPR/Cas9 have already resulted in successful FDA approval for cell-based gene editing therapies, providing new therapeutic avenues for many rare diseases. However, immune responses to gene therapy delivery vectors and editing tools remain a challenge, especially for strategies targeting complex in vivo tissues such as the lung. Previous findings in non-CF healthy individuals reported pre-existing antibody and T cell dependent immune responses to recombinant Cas9 proteins, suggesting potential additional obstacles for incorporation of CRISPR/Cas9 technologies in gene therapies. To determine if pre-existing immunity to Cas9 from S. aureus or S. pyogenes was present or augmented in people with CF (PwCF), anti-Cas9 IgG levels and Cas9-specific T cell responses were determined from peripheral blood samples of PwCF and non-CF healthy controls. Overall, non-CF controls and PwCF displayed evidence of pre-existing antibody and T cell responses to both S. aureus and S. pyogenes Cas9, although there were no significant differences between the two populations. However, we observed global changes in activation of Th1 and CD8 T cell responses as measured by IFNγ and TNF that warrant further investigation and mechanistic understanding as this finding has implications not only for CRISPR/Cas9 gene therapy for PwCF, but also for protection against infectious disease.

Although two-thirds of cancers arise from loss-of-function mutations in tumor suppressor genes, there are few approved targeted therapies linked to these alterations. Synthetic lethality offers a promising strategy to treat such cancers by targeting vulnerabilities unique to cancer cells with these mutations. To identify clinically relevant synthetic lethal interactions, we analyzed genome-wide CRISPR/Cas9 knock-out (KO) viability screens from the Cancer Dependency Map and evaluated their clinical relevance in patient tumors through mutual exclusivity, a pattern indicative of synthetic lethality. Indeed, we found significant enrichment of mutual exclusivity for interactions involving cancer driver genes compared to non-driver mutations. To identify therapeutic opportunities, we integrated drug sensitivity data to identify inhibitors that mimic the effects of CRISPR-mediated KO. This approach revealed potential drug repurposing opportunities, including BRD2 inhibitors for bladder cancers with ARID1A mutations and SIN3A-mutated cell lines showing sensitivity to nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. However, we discovered that pharmacological inhibitors often fail to phenocopy KO of matched drug targets, with only a small fraction of drugs inducing similar effects. This discrepancy reveals fundamental differences between pharmacological and genetic perturbations, emphasizing the need for approaches that directly assess the interplay of loss-of-function mutations and drug activity in cancer models.

RNA polymerase II (Pol II) facilitates co-transcriptional splicing by recruiting the U1 small nuclear ribonucleoprotein particle (U1 snRNP) to the nascent transcripts. Here, we report the cryo-electron microscopy structure of a transcribing Pol II-U1 snRNP complex with elongation factors DSIF and SPT6. Furthermore, our biochemical analysis revealed that the phosphorylated Pol II carboxyl-terminal domain and SPT6 interact directly with U1 snRNP proteins, facilitating its recruitment to the elongation complex. This multivalent interaction allows efficient spliceosome assembly and ensures transcription processivity.

Although centrioles and primary cilia play an essential role in early mammalian development, their specific function during the interval between their initial formation and the subsequent arrest of embryogenesis in embryos deficient in centrioles or cilia remains largely unexplored. Here, we demonstrate that different 3D in vitro model systems recapitulate early centriole and cilium formation in mouse development. Centrioles and cilia are dispensable in 3D in vitro mouse rosettes, a model system that mimics key events of implantation, including polarization and lumenogenesis. In gastruloids, a model system that recapitulates developmental processes up to 8.5 days after fertilization, centriole loss results in early disassembly. In contrast, cells devoid of cilia continue to form elongated, differentiated and polarized gastruloids, with minor differences at 96 h. Finally, we show that in a mutant affecting the centriolar distal appendages, cilia are absent from 2D cultures but are capable of forming in 3D rosettes and gastruloids, highlighting the importance of multifactorial 3D environment setups in developmental studies. Summary This study presents the first in vitro analysis of centriole and cilium formation during early mouse embryonic development, using 3D models to mimic implantation, tissue patterning, and axis elongation, offering a controlled platform for investigating their roles in embryogenesis.

Abstract Sphingolipids play crucial roles in cell membrane structure and in multiple signaling pathways. Sphingolipid de novo biosynthesis is mediated by the serine palmitoyltransferase (SPT) enzyme complex. Homeostatic regulation of this complex is dependent on its regulatory subunit, the ORMDLs, of which there are three isoforms. It is well established that the ORMDLs regulate SPT activity, but it is still unclear whether the three ORMDL isoforms have distinct functions and properties. Here, we focus on understanding the physiological importance of ORMDL isoforms (ORMDL1, ORMDL2, and ORMDL3) in regulating SPT activity and sphingolipid levels. This study delves into the differential responses of the SPT complexes containing different ORMDL isoforms to cellular ceramide levels. By using the CRISPR/Cas9 gene editing tool, we have developed Hela cell lines each of which harbor only one of the three ORMDL isoforms as well as a cell line deleted for all three isoforms. Consistent with other studies, we find that deletion of all three ORMDL isoforms desensitizes SPT to ceramide and dramatically increases levels of cellular sphingolipids. In contrast, each ORMDL isoform alone is capable of regulating SPT activity and maintaining normal levels of sphingolipid. Strikingly, however, we find that each ORMDL isoform exhibits isoform-specific sensitivity to ceramide. This suggests that the inclusion of specific ORMDL isoforms into the SPT complex may accomplish a fine-tuning of sphingolipid homeostasis. The study not only emphasizes the need for further investigation into the distinct roles of ORMDL isoforms but also sheds light on their potential as therapeutic targets. Highlights RMDL isoforms detect varying ceramide levels to regulate SPT. HeLa cells, there is no compensation for the absence of the ORMDL isoform, neither at the total protein level nor at the mRNA level.

Transcription of protein coding genes in trypanosomatids is atypical and almost exclusively polycistronic. In Trypanosoma brucei , approximately 150 polycistrons, and 8000 genes, are constitutively transcribed by RNA polymerase II. RNA polymerase II promoters are unconventional and characterised by regions of chromatin enriched for histones with specific patterns of post-translational modification on their highly divergent N-terminal tails. To investigate the roles of histone tail-residues in gene expression control in T. brucei , we engineered strains exclusively expressing novel mutant histones. We used an inducible CRISPR-Cas9 system to delete >40 native copies of histone H4 , complementing the tandem arrays with a single ectopic H4 gene. The resulting ‘hist one H4’ strains were validated using whole-genome sequencing and transcriptome analysis. We then performed saturation mutagenesis of six histone H4 N-terminal tail lysine (K) residues and used multiplex amplicon-seq to profile the relative fitness of 384 distinct precision edited mutants. H4 K10 mutations were not tolerated, but we could derive a panel of nineteen strains exclusively expressing novel H4 K4 or H4 K14 mutants. Both proteomic and transcriptomic analysis of H4 K4Q mutants revealed significantly reduced expression of genes adjacent to RNA polymerase II promoters, where the glutamine (Q) mutation mimics an abnormally high level of acetylation. Thus, we present direct evidence for polycistronic expression control by histone H4 N-terminal tails in trypanosomes.

Abstract Background: The development of functional muscles in Drosophila melanogaster relies on precise spatial and temporal transcriptional control, orchestrated by complex gene regulatory networks. Central to this regulation are cis-regulatory modules (CRMs), which integrate inputs from transcription factors to fine-tune gene expression during myogenesis. In this study, we investigate the transcriptional regulation of the LIM-homeodomain transcription factor Tup (Tailup/Islet-1), a key regulator of dorsal muscle development. Methods: Using a combination of CRISPR-Cas9-mediated deletion and transcriptional analyses, we examined the role of multiple CRMs in regulating tup expression. Results: We demonstrate that tup expression is controlled by multiple CRMs that function redundantly to maintain robust tup transcription in dorsal muscles. These mesodermal tup CRMs act sequentially and differentially during the development of dorsal muscles and other tissues, including heart cells and alary muscles. We show that activity of the two late-acting CRMs govern late-phase tup expression through positive autoregulation, whereas an early enhancer initiates transcription independently. Deletion of both late-acting CRMs results in muscle identity shifts and defective muscle patterning. Detailed morphological analyses reveal muscle misalignments at intersegmental borders. Conclusions: Our findings underscore the importance of CRM-mediated autoregulation and redundancy in ensuring robust and precise tup expression during muscle development. These results provide insights into how multiple CRMs coordinate gene regulation to ensure proper muscle identity and function.

A cell’s fate is shaped by its inherited state, or lineage, and the ever-shifting context of its environment. CRISPR-based recording technologies are a promising solution to map the lineage of a developing system, yet challenges remain regarding single-cell recovery, engineering complexity, and scale. Here, we introduce BASELINE, which uses base editing to generate high-resolution lineage trees in conjunction with single-cell profiling. BASELINE uses the Cas12a adenine base editor to irreversibly edit nucleotides within 50 synthetic target sites, which are integrated multiple times into a cell’s genome. We show that BASELINE accumulates lineage-specific marks over a wide range of biologically relevant intervals, recording more than 4300 bits of information in a model of pancreatic cancer, a 50X increase over existing technologies. Single-cell sequencing reveals high-fidelity capture of these recorders, recovering lineage reconstructions up to 40 cell divisions deep, within the estimated range of mammalian development. We expect BASELINE to apply to a wide range of lineage-tracing projects in development and disease, especially in which cellular engineering makes small, more distributed systems challenging.

Patterning of the neural tube establishes midbrain and hindbrain structures that coordinate motor movement, process sensory input, and integrate cognitive functions. Cellular impairment within these structures underlie diverse neurological disorders, and in vitro organoid models promise inroads to understand development, model disease, and assess therapeutics. Here, we use paired single-cell transcriptome and accessible chromatin sequencing to map cell composition and regulatory mechanisms in organoid models of midbrain and hindbrain. We find that existing midbrain organoid protocols generate ventral and dorsal cell types, and cover regions including floor plate, dorsal and ventral midbrain, as well as adjacent hindbrain regions, such as cerebellum. Gene regulatory network (GRN) inference and transcription factor perturbation resolve mechanisms underlying neuronal differentiation. A single-cell multiplexed patterning screen identifies morphogen concentration and combinations that expand existing organoid models, including conditions that generate medulla glycinergic neurons and cerebellum glutamatergic subtypes. Differential abundance of cell states across screen conditions enables differentiation trajectory reconstruction from region-specific progenitors towards diverse neuron types of mid- and hindbrain, which reveals morphogen-regulon regulatory relationships underlying neuronal fate specification. Altogether, we present a single-cell multi-omic atlas and morphogen screen of human neural organoid models of the posterior brain, advancing our understanding of the co-developmental dynamics of regions within the developing human brain.

Background Calcium ions (Ca²⁺) serve as universal intracellular messengers regulating diverse physiological processes, while dysregulated Ca²⁺ homeostasis triggers cytotoxicity. Molecular hydrogen (H₂) exhibits protective effects against oxidative stress-related pathologies, but its mechanism of action remains incompletely understood. Transient receptor potential canonical 4 (TRPC4) channels and their associated protein TRPC4AP are critical mediators of Ca²⁺ influx ([Ca²⁺]i), yet their role in H₂-mediated calcium signaling is unexplored. This study investigates the molecular mechanism by which H₂ modulates Ca²⁺ dynamics through the TRPC4-TRPC4AP axis, aiming to establish its therapeutic potential for calcium-related disorders. Methods The study employed heterogeneous cellular models (e.g., mesenchymal stem cells, neurons, fibroblasts) and in vivo two-photon calcium imaging in C57BL/6J mice. Techniques included CRISPR-Cas9 knockout, siRNA-mediated gene silencing, molecular docking (AlphaFold 3), and protein-protein interaction analysis. Calcium flux was quantified via fluorescence imaging, while mitochondrial integrity and cytoskeletal dynamics were assessed using JC-1 staining, ATPase activity assays, and live-cell imaging. Structural validation of TRPC4-TRPC4AP binding sites utilized mutagenesis and complementation experiments. Results H₂ selectively enhanced extracellular Ca²⁺ influx via TRPC4-TRPC4AP, with no cytotoxicity or mitochondrial dysfunction observed. Key arginine residues (730Arg-731Arg) in the TRPC4 CIRB domain formed hydrogen-bond networks essential for channel activation. In vivo, H₂ increased neuronal Ca²⁺ transient frequency and amplitude in the primary motor cortex. TRPC4AP knockout abolished H₂-induced Ca²⁺ influx, while mutagenesis of 730Arg/731Arg disrupted channel activity. H₂ also promoted cytoskeletal remodeling and cell motility, dependent on TRPC4AP-mediated Ca²⁺ signaling. Conclusions This study identifies H₂ as a novel calcium agonist that activates the TRPC4-TRPC4AP axis to regulate extracellular Ca²⁺ influx. The 730Arg-731Arg motif in TRPC4 serves as a critical H₂-sensitive site, enabling dynamic calcium homeostasis without overload. These findings provide a mechanistic basis for H₂-based therapies targeting calcium dysregulation in neurodegenerative, inflammatory, and metabolic diseases, while highlighting TRPC4AP as a pivotal molecular switch for gasotransmitter signaling.

SUMMARY Translocating unfolded polypeptides across membranes is essential in all domains of life and in bacteria requires the conserved Sec machinery and ATP. Bacterial Sec substrates fold outside the cell and often use DsbA-catalysed disulfide bond formation between cysteines to ensure correct folding. Extracellular protein misfolding triggers a stress response that involves production of dual function HtrA-family chaperone/proteases. In Gram-negative bacteria this is called the envelope stress response and in Gram-positive bacteria the secretion stress response, but the exact signals sensed by bacteria to trigger these stress responses are not well understood. In Streptomyces bacteria the secretion stress response is mediated by the CutRS and CssRS two-component systems which control the levels of four conserved HtrA-family chaperones. Here we show that the CutS sensor kinase contains two conserved cysteine residues in its extracellular sensor domain that control CutS activity. CssS also has two conserved and invariant cysteines in its sensor domain, and we propose that CutS and CssS detect the extracellular redox state and work together to ensure secreted proteins fold correctly in the fluctuating soil environment. Further analysis of ∼12,800 genomes indicated that 98.9% of strains across all bacterial classes have at least one sensor kinase with two or more extracellular cysteine residues, suggesting that extracellular redox sensing by two-component systems is widespread in bacteria.

Targeted gene editing can be achieved using CRISPR/Cas9-assisted recombineering. However, high-efficiency editing requires careful optimization for each locus to be modified which can be tedious and time-consuming. In this work, we developed a simple, fast and cheap method for the E diting and A ssembly of SY nthetic operons using CRISPR/Cas9-assisted recombineering (EASY-CRISPR) in Escherichia coli . Highly efficient editing of the different constitutive elements of the operons can be achieved by using a set of optimized guide RNAs and single- or double-stranded DNA repair templates carrying relatively short homology arms. This facilitates the construction of multiple genetic tools, including mutant libraries or reporter genes. EASY-CRISPR is also highly modular, as we provide alternative and complementary versions of the operon inserted in three loci which can be edited iteratively and easily combined. As a proof of concept, we report the construction of several fusions with reporter genes confirming known post-transcriptional regulation mechanisms and the construction of saturated and unbiased mutant libraries. In summary, the EASY-CRISPR system provides a flexible genomic expression platform that can be used both for the understanding of biological processes and as a tool for bioengineering applications.

Abstract Saffold virus (SAFV), a member of the species Cardiovirus saffoldi within the Picornaviridae family, causes acute respiratory and gastrointestinal illnesses, as well as hand, foot, and mouth diseases. It is also suspected to be associated with neuronal disorders such as encephalitis and meningitis in severe cases. Despite its clinical significance, the virus-host interactions underlying SAFV pathogenicity remain largely unknown. Using a genome-wide CRISPR-Cas9 knockout screen, we identified receptors for SAFV infection: sulfated glycosaminoglycans (GAGs) and integrin aVb8. Single knockouts of SLC35B2 , an essential gene for sulfated GAG synthesis, or the integrin genes, ITGAV or ITGB8 partially reduced SAFV-3 susceptibility in HeLa cells, and double knockout conferred complete resistance. Furthermore, we demonstrated that SAFV-3 virions bind directly to sulfated GAGs and integrin aVb8. Based on these findings, we propose a model of SAFV infection, in which sulfated GAGs and integrin aVb8 function in parallel pathways during viral entry.

Abstract Duchenne muscular dystrophy (DMD) is a severe X-linked disorder caused by mutations in the DMD gene, with a global prevalence of 3.6 per 100,000 people. Despite its well-documented genetic basis, no previous studies have characterised DMD in Guatemala. We analysed 33 genetically confirmed cases to estimate prevalence, describe the mutation spectrum, and assess clinical features. Prevalence was 0.61 per 100,000 men under 30. Symptoms began before age 5 in 85% of cases, yet 60% were diagnosed after age 6, highlighting significant diagnostic delays. Deletions were the most common mutation (55%), followed by point mutations (30%) and duplications (15%), with two novel variants identified. Most deletions clustered in the exon 45–55 hotspot. Nearly half of the cases were eligible for exon-skipping therapies. These findings reveal genetic heterogeneity in the Guatemalan population, substantial delays in diagnosis, and the need for improved access to genetic testing, targeted treatments, and a national DMD registry.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels and the main mediators of synaptic neurotransmission in the insect brain. In insects, nAChRs are pivotal for sensory processing, cognition and motor control, and are the primary target of neonicotinoid insecticides. Neonicotinoids are potent neurotoxins and pollinators such as honey bees are more sensitive and affected by extremely low sub-lethal doses. The pentameric nAChR channel is made up either of five α-subunits constituting five ligand-binding sites or a mixture of two to three α and β subunits constitute two to three ligand-binding sites. Of particular note, the honey bee nAChRα8 subunit is converted into a β subunit (nAChRβ2) in Drosophila , raising the question whether this α to β conversion makes flies less sensitive to neonicotinoids. To investigate species-specific aspects of neonicotinoid toxicity we CRISPR-Cas9 engineered a cross-species chimeric nAChR subunit by swapping the ligand-binding domain in Drosophila of nAChRβ2 with honey bee nAChRα8. Toxicity assessment by neonicotinoid thiamethoxam revealed significantly impaired motor functions in climbing and flight assays when comparing the α8/β2 chimeric channel to wild type or a β2 knock-out. However, both the α8/β2 chimeric channel and the β2 knock-out showed the same increased survival after neonicotinoid exposure compared to wild type flies. Combinatorial exposure to neonicotinoids also did not reveal differences. These findings highlight the critical role of nAChR subunit composition in motor control and demonstrate how subtle structural differences can profoundly impact motor function and pesticide response, offering new insights into the molecular mechanisms of neurotoxicity across species.

Reactive oxygen species (ROS) pose a significant threat to biological molecules, prompting organisms to develop systems that buffer oxidative stress and contain iron, which otherwise amplifies ROS production. Understanding oxidative stress responses requires identifying the key proteins involved and their cellular organization. Here, we combined proteomics and cryo-EM to investigate the response of the anaerobic hyperthermophilic archaeon Pyrococcus furiosus to oxygen exposure. Proteome analysis revealed a significant upregulation of the oxidoreductase Rubrerythrin (Rbr) under oxidative stress. Cryo-electron tomograms showed the formation of prominent oxidative stress-induced tubules (OSITs). Single-particle cryo-EM and mass spectrometry of enriched OSITs identified them as stacked rings of Rbr homotetramers. The 3.3 Å structure demonstrates that rubredoxin-like domains mediate homotetramer assembly, suggesting that their oxidation drives OSIT formation. Within OSITs, we discovered virus-like particles formed by a ferritin-like/encapsulin fusion protein with iron hydroxide cores, uncovering a sophisticated organelle that protects P. furiosus from ROS through advanced compartmentalization.

SUMMARY Natural killer (NK) cells, a type of potent cytotoxic lymphocytes, are particularly promising for the treatment of cancers that lose or downregulate major histocompatibility complex class I (MHC-I) expression to evade T cell-mediated immunotherapy. However, the hostile and immune suppressive tumor microenvironment (TME) greatly hinders the function of tumor-infiltrating NK cells limiting the therapeutic efficacy against solid tumors. Here, we show that a fusion protein of interleukin-21 (IL-21−Fc), as a direct in vivo intervention, can safely and effectively reprogram NK cell metabolism and enhance their effector function in the TME. Our research demonstrates that combining IL-21−Fc with IL-15 superagonist (IL-15SA) or NK cell transfer leads to the eradication of MHC-I-deficient tumors and confers durable protection in syngeneic and xenograft tumor models. Mechanistically, we uncover that IL-21−Fc enhances NK cell effector function by upregulating glycolysis in a lactate dehydrogenase A (LDHA)-dependent manner. These findings not only underscore the considerable potential of IL-21−Fc as an in vivo therapeutic intervention to bolster NK cell-based immunotherapy, but also unveil an innovative strategy of metabolic reprogramming for NK cell rejuvenation within tumors. GRAPHICAL ABSTRACT HIGHLIGHTS NK cells display functional exhaustion in MHC-I deficient solid tumors IL-21−Fc as an in vivo-applicable and safe immunotherapy reinvigorates intratumoral NK cells for enhanced effector function IL-21−Fc enhances NK cell function by elevating glycolysis in a LDHA-dependent manner Combining IL-21−Fc with low-dose IL-15SA or NK cell transfer eradicates syngeneic and xenografted solid tumors

Abstract RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse CRISPR-Cas effectors, CRISPR-Casλ, a recently identified miniature type V effector encoded in phage genomes, has emerged as a promising candidate for genome editing due to its nuclease activity in mammalian and plant cells. However, the detailed molecular mechanisms of Casλ family of enzymes remain poorly understood. In this study, we report the identification and detailed biochemical and structural characterizations of CRISPR-Casλ2. The cryo-electron microscopy structures of Casλ2 in five different functional states unveiled the dynamic domain rearrangements during its activation. The structures revealed that, unlike other type V CRISPR-Cas effectors, the REC2 domain directly interacts with the substrate DNA within the RuvC active site to facilitate the target DNA cleavage. Our biochemical analyses indicated that Casλ2 processes its precursor crRNA to a mature crRNA using the RuvC active site through a unique ruler mechanism, in which Casλ2 defines the spacer length of the mature crRNA. Furthermore, structural comparisons of Casλ2 with Casλ1 and CasΦ highlighted the diversity and conservation of phage-encoded type V CRISPR-Cas enzymes. Collectively, our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and establish a framework for rational engineering of the CRISPR-Casλ-based genome-editing platform.

Stone fruits (Prunus spp.) occupy a pivotal position in global fruit production due to their significant nutritional profile and distinctive organoleptic characteristics. Contemporary orchard systems are undergoing transformation through innovative cultivation approaches, notably high-density dwarfing systems, greenhouse cultivation, agri-tech integration, and simplified management. As a crucial agronomic component in modern stone fruit cultivation, rootstock systems confer multi-benefits including enhanced environmental resilience, improved scion productivity, superior fruit quality, controlled vigor and dwarfing capacity. While the majority of european apple orchards have transitioned to dwarfing rootstock systems, achieving substantial gains in productivity and profitability, stone fruits cultivation lag significantly due to the key gaps in prunus rootstock development include genetic complexity, extended evaluation cycles, clonal propagation barriers and limited research programs. Urgent innovation is required to address these challenges in rootstock breeding to meet the demand of sustainable stone fruits production. This review systematically examines strategic breeding objectives and innovative molecular methodologies in Prunus rootstock development, with particular emphasis on marker-assisted selection and genomic prediction technologies. We provide a comprehensive synthesis of breeding achievements across major commercial rootstock cultivars, while proposing forward-looking research strategies incorporating CRISPR-based genome editing and multi-omics approaches. The synthesized insights establish a theoretical pathway for advancing rootstock genetic improvement and sustainable orchard management practices in stone fruit cultivation systems.

Sotorasib (AMG510) and adagrasib (MRTX849) have shown significant efficacy in KRAS G12C mutant NSCLC, but acquired resistance occurs within 6–12 months. While some resistance arises from new mutations, over half of the resistant cases lack identifiable genomic alterations. We hypothesize that resistance is driven by signaling network rewiring, creating new therapeutic vulnerabilities. To investigate acquired resistance (AR) mechanisms, multiple AR models, including cell lines (H23AR & H358AR), PDXs (TC303AR & TC314AR), CDXs (H358AR CDX), and PDXOs (PDXO303AR & PDXO314AR) were developed. H23AR and H358AR cells displayed >600-fold and 200-fold and PDXO303AR and PDXO314AR, exhibited >300-fold and >100-fold resistance to sotorasib, respectively compared to their parental counterparts, however, no additional mutations in KRAS or other potential genetic alterations were identified. The AR cells and PDXOs also showed comparable resistance to adagrasib. Proteomic and phosphoproteomic analyses in TC303AR & TC314AR PDXs identified distinct protein signatures associated with KRAS reactivation, mTORC1 signaling upregulation, and PI3K/AKT/mTOR pathway activation. PI3K protein levels were significantly elevated in AR PDXs, H23AR, and H358AR cells. Pharmacological inhibition of PI3K with copanlisib or genetic knockout via CRISPR-Cas9 restored sotorasib sensitivity, suppressed colony formation, and inhibited downstream effectors, including p-AKT, p-mTOR, p-S6, p70S6K, p-GSK3β, and p-PRAS40 in AR cells. copanlisib also sensitized both acquired and primary resistant PDXOs and synergized with sotorasib in restoring drug sensitivity. We found that p4E-BP1 was significantly upregulated in H23AR and H358AR cells, and copanlisib suppressed its expression. The level of p4E-BP1 expression was correlated with Sotorasib sensitivity in PI3K knockout clones, where the most sensitive clone displayed reduced or no p4E-BP1 expression. CRISPR-Cas9-mediated knockout of 4E-BP1, either alone or in combination with PI3K knockout, dramatically restored sotorasib sensitivity to levels comparable to parental cells. Suppression of 4E-BP1 hyperphosphorylation required dual inhibition of mTORC1 and mTORC2, and treatment with AZD8055 or sapanisertib (mTORC1/2 dual inhibitors) significantly dephosphorylated 4E-BP1 and restored sotorasib sensitivity in resistant cells and PDXOs. In contrast, everolimus (a mTORC1-selective inhibitor) did not restore sotorasib sensitivity. In PDX, CDX, and xenograft models in vivo, the combination of sotorasib with either copanlisib or sapanisertib resulted in robust, synergistic, and durable tumor regression at well-tolerated doses. These findings showed the critical role of PI3K/mTOR signaling as a bypass mechanism of resistance to KRAS G12C inhibitors. We conclude that mTORC1/2 mediated inhibition of p4E-BP1 and combination strategies targeting this pathway effectively overcomes acquired resistance to KRAS G12C inhibitors in NSCLC.