CRISPR homing drives can be used to suppress a population by targeting female fertility genes. They convert wild-type alleles to drive alleles in the germline of drive heterozygotes by homology-directed repair after DNA cleavage. However, resistance alleles produced by end-joining pose a great threat to homing drive. They prevent further recognition by Cas9, and therefore weaken suppressive power, or even stop suppression if they preserve the function of the target gene. We used multiplexed gRNAs targeting doublesex in Drosophila to avoid functional resistance and create resistance alleles that were dominant female-sterile. This occurred because the male dsx transcript was generated in females by disruption of the female-specific splicing acceptor site. We rescued dominant sterility of the drive by providing an alternate splicing site. As desired, the drive was recessive female sterile and yielded high drive inheritance among the progeny of both male and female drive heterozygotes. The dominant-sterile resistance alleles enabled stronger suppression in computational models, even in the face of modest drive efficiency and fitness costs. However, we found that male drive homozygotes were also sterile because they used the rescue splice site. Attempts to rescue males with alternate expression arrangements were not successful, though some male homozygotes had less severe intersex phenotypes. Though this negatively impacted the drive, models showed that it still had significantly improved suppressive power. Therefore, this design may have wide applicability to dsx -based suppression gene drives in a variety of organisms with intermediate homing drive performance.

Multi-trait QTL (xQTL) colocalization has shown great promises in identifying causal variants with shared genetic etiology across multiple molecular modalities, contexts, and complex diseases. However, the lack of scalable and efficient methods to integrate large-scale multi-omics data limits deeper insights into xQTL regulation. Here, we propose ColocBoost , a multi-task learning colocalization method that can scale to hundreds of traits, while accounting for multiple causal variants within a genomic region of interest. ColocBoost employs a specialized gradient boosting framework that can adaptively couple colocalized traits while performing causal variant selection, thereby enhancing the detection of weaker shared signals compared to existing pairwise and multi-trait colocalization methods. We applied ColocBoost genome-wide to 17 gene-level single-nucleus and bulk xQTL data from the aging brain cortex of ROSMAP individuals (average N = 595), encompassing 6 cell types, 3 brain regions and 3 molecular modalities (expression, splicing, and protein abundance). Across molecular xQTLs, ColocBoost identified 16,503 distinct colocalization events, exhibiting 10.7(± 0.74)-fold enrichment for heritability across 57 complex diseases/traits and showing strong concordance with element-gene pairs validated by CRISPR screening assays. When colocalized against Alzheimer’s disease (AD) GWAS, ColocBoost identified up to 2.5-fold more distinct colocalized loci, explaining twice the AD disease heritability compared to fine-mapping without xQTL integration. This improvement is largely attributable to ColocBoost ’s enhanced sensitivity in detecting gene-distal colocalizations, as supported by strong concordance with known enhancer-gene links, highlighting its ability to identify biologically plausible AD susceptibility loci with underlying regulatory mechanisms. Notably, several genes including BLNK and CTSH showed sub-threshold associations in GWAS, but were identified through multi-omics colocalizations which provide new functional support for their involvement in AD pathogenesis.

Neural stem cell (NSC) transplantation is a promising therapeutic approach for spinal cord repair, but poor graft survival remains a critical challenge. Here, we demonstrate that the mechanical properties of the transplantation microenvironment play a crucial role in NSC survival in the injured spinal cord. While our previously engineered imidazole-poly(organophosphazene) (I-5) hydrogel effectively prevented cavity formation by promoting extracellular matrix remodeling, NSCs transplanted with 10% hydrogel exhibited poor survival. Remarkably, increasing the hydrogel concentration to 16%, which created a 5-fold stiffer matrix, significantly enhanced NSC graft survival and synaptic integration. Using in vitro models with controlled substrate stiffness, we found that NSCs on stiffer substrates displayed enhanced adhesion, complex morphology, and increased viability. Importantly, we identified the mechanosensitive ion channel Piezo1 as the key molecular mediator of these stiffness-dependent behaviors. CRISPR/Cas9-mediated Piezo1 gene editing in NSCs significantly reduced graft survival in vivo when transplanted with 16% hydrogel, confirming that Piezo1-mediated mechanotransduction is essential for NSC survival in the injured spinal cord. Our findings reveal a previously unrecognized mechanism governing graft survival in the injured spinal cord and suggest that optimizing the mechanical properties of biomaterial scaffolds or targeting Piezo1-dependent mechanotransduction could substantially improve outcomes of cell-based therapies for neurological disorders.

Summary We investigated the roles of Rac guanine-nucleotide factor (Rac-GEF) Prex1 in glucose homeostasis using Prex1 −/− and catalytically-inactive Prex1 GD mice. Prex1 maintains fasting blood glucose levels and insulin sensitivity through its Rac-GEF activity but limits glucose clearance independently of its catalytic activity, throughout ageing. Prex1 −/− mice on high-fat diet are protected from developing diabetes. The increased glucose clearance in Prex1 −/− mice stems from constitutively enhanced hepatic glucose uptake. Prex1 limits Glut2 surface levels, mitochondrial membrane potential and mitochondrial ATP production, and controls mitochondrial morphology in hepatocytes, independently of its catalytic activity. Prex1 limits GPCR trafficking through an adaptor function, and we identify here the inhibitory orphan GPCR Gpr21 as a Prex1 target. The Gpr21-mediated blockade of glucose uptake and mitochondrial ATP production in hepatocytes requires Prex1. We propose that Prex1 limits glucose clearance by maintaining Gpr21 at the hepatocyte surface, thus limiting hepatic glucose uptake and metabolism. Graphical abstract

Intermittent fasting and fasting-refeeding regimens can slow biological aging across taxa 1 . Shifts between fed and fasted states activate ancient nutrient-sensing pathways which alter cellular and epigenetic states to promote longevity 2–4 . Yet how biological age trajectories progress during fasting-refeeding, and how nutrient-sensing pathways reprogram epigenetic state remain largely unknown. Here we observe increases in predicted biological age of Caenorhabditis elegans during prolonged fasting in adult reproductive diapause, followed by extraordinary reduction of biological age during refeeding. We identify hil-1 / H1-0 as an evolutionarily conserved nutrient-regulated linker histone which mediates adaptations to fasting and refeeding downstream of FOXO and TFEB transcription factors. In C. elegans and human cell culture, hil-1 / H1-0 upregulation during low-nutrient states promotes long-term survival and subsequent refeeding-induced recovery. Restoration of C. elegans after prolonged fasting is improved by enhancing the natural downregulation of hil-1 specifically during refeeding. Our study identifies HIL-1/H1.0 as part of an ancestral epigenetic switch during fasting-refeeding that reprograms metabolic and cellular states underlying resilience and restoration.

Accumulation of misfolded α-synuclein protein in intracellular inclusion bodies of dopaminergic neurons underlies the pathogenesis of Synucleinopathies, which include Parkinson’s Disease (PD), Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA). Therefore, clearance of misfolded α-synuclein from dopaminergic neurons could in principle offer a therapeutic window for Synucleinopathies, which currently remain untreatable. In this study, we employ the Affinity-directed PROtein Missile (AdPROM) system consisting of the substrate receptor of the CUL2-E3 ligase complex VHL and a nanobody selectively recognising the human α-synuclein protein and demonstrate targeted degradation of endogenous α-synuclein from human cell lines with remarkable selectivity. We further demonstrate that targeted degradation of α-synuclein prevents the pre-formed fibril (PFF)-induced aggregation of α-synuclein in primary neurons derived from rats expressing human α-synuclein. This approach represents the first demonstration of nanobody-guided proteasomal degradation of all clinically relevant α-synuclein variants, highlighting its potential as a therapeutic strategy against Synucleinopathies.

Leukaemias, driven by mutations in hematopoietic stem cells (HSCs), rely on interactions with the bone marrow (BM) niche and other cell populations such as mesenchymal stromal cells (MSCs) for growth and survival. While chimeric antigen receptor (CAR) T-cell therapy shows promise for other hematological malignancies, its application to acute myeloid leukaemia (AML) is hindered by tumour heterogeneity and off-target toxicity. Combining CRISPR-Cas9 gene editing with CAR T-cell therapy has potential for selectively targeting AML cells while sparing healthy tissue. However, validating the efficacy of these treatments prior to clinical trial is hampered by the differences between humans and animal models typically used for pre-clinical testing. Furthermore, traditional in vitro models fail to replicate the complexity of the BM niche and often overestimate treatments’ efficacy. Here, we present a bioengineered human-cell containing BM niche model combining a fibronectin-presenting polymeric surface and a synthetic peptide hydrogel (PeptiGel) that mimics native BM tissue’s mechanical properties. This platform supports niche phenotypes in MSCs and HSCs and enables the evaluation of combined CRISPR-CAR T-cell therapy, demonstrating potential as a preclinical human model for testing novel therapies.

Epithelial cancers such as stomach and ovary cancer tend to metastasize to the peritoneum, often leading to intractable disease and poor survival. Currently, the mechanisms that enable gastric cancer cells to penetrate the mesothelium, and to implant, invade, and survive in the peritoneal niche are poorly understood. To investigate these mechanisms, we developed a novel human peritoneal explant model. Briefly, fresh peritoneal tissue samples from abdominal surgery patients were cultured on top of a layer of GFP-labeled human gastric adenocarcinoma cells (AGS); 2% of these cells implanted into the peritoneum. The transcriptomic profile of the implanted AGS cells was compared to the profile of AGS cells that failed to implant using RNA sequencing. Differentially expressed genes were enriched significantly (fold change>2) for genes that enable cell adhesion, motility, and membrane depolarization. We compared this list of genes with a previously identified peritoneal metastasis whole exome sequencing dataset (SRP043661). Upon further analysis based on subcellular localization, cell adhesion and cytoskeletal organization, we found nine core “peritoneal implantation” genes. We functionally validated these genes with CRISPR knockout and assessed peritoneal implantation and invasion using the human peritoneal explant model described above. From these data we identified ADAM12 as a key player of peritoneal metastasis. Knock out of ADAM12 significantly impaired peritoneal metastasis in vivo and ex vivo . Exploration of three publicly available independent datasets indicated that ADAM12 is indeed clinically relevant in peritoneal metastasis. To explore the role of ITGAβ1 in mediating cell-matrix interactions in the presence of ADAM12, we performed ITGAβ1 pull-down assays followed by mass spectrometry analysis in ADAM12 WT cells. ADAM12 KO cells show a marked disruption of the ITGAβ1 interactome in GCa cells. Key cytoskeletal proteins such as MYH14, MYH10, MYH9, ACTA2, SPTN1 and TPM1–TPM4 were found to be interactors with ITGAβ1 in ADAM12 WT cells but not ADAM12 KO cells. Our approach and the new data identify a distinct peritoneal metastasis gene set that facilitates implantation and invasion of gastric cancer cells within the peritoneum. Disruption of these pathways with peritoneal-directed therapies has the potential to improve survival in patients with high-risk primary gastric cancer.

Summary Cancer immunotherapy is only effective in a subset of patients, highlighting the need for effective biomarkers and combination therapies. Here we systematically identify genetic determinants of cancer cell sensitivity to anti-tumor immunity by performing whole-genome CRISPR/Cas9 knock-out screens in autologous tumoroid-T cell co-cultures, isogenic cancer cell models deficient in interferon signaling, and in the context of four cytokines. We discover that loss of CHD1 and MAP3K7 potentiates the transcriptional response to IFN-γ, thereby creating an acquired vulnerability through sensitizing cancer cells to tumor-reactive T cells. Immune checkpoint blockade was more effective in a syngeneic mouse model of melanoma deficient in Chd1 and Map3k7 and was associated with elevated intra-tumoral CD8 + T cell numbers and activation. CHD1 and MAP3K7 are recurrently mutated in cancer and reduced expression in tumors correlates with response to immune checkpoint inhibitors in patients, nominating these genes as potential biomarkers of immunotherapy response.

ABSTRACT The development of traditional protein-targeted cancer therapies is a slow and arduous process, often taking years or even decades. In contrast, RNA-based therapies targeting crucial microRNAs (miRNAs) offer a faster alternative due to the sequence specific nature of miRNA inhibitor binding. This, combined with the capacity of individual miRNAs to influence multiple cellular pathways, makes these small RNAs attractive targets for cancer therapy. While miRNA are known to be dysregulated in prostate cancer (PCa), identifying their individual contributions to disease progression and the identification of therapeutically actionable miRNA targets in PCa has been challenging due to limited screening tools. To overcome this, we developed miRKOv2, a miRNA-only CRISPR knockout library enabling systematic, genome-wide loss-of-function screens to identify miRNAs essential for PCa cell survival. Our screens uncovered 69 potential essential miRNA candidates, with miR-483 demonstrating the most significant impact on PCa cell viability. Functional characterization demonstrated that miR-483 disruption significantly potentiated apoptosis in PCa cell lines. Mechanistically, we uncovered a novel regulatory axis wherein miR-483-3p directly modulates a BCLAF1/PUMA/BAK1 apoptotic signaling network, highlighting its critical role in maintaining PCa cell survival. Our findings provide novel insights into the complex regulatory role of miRNA in PCa progression and offer a potential therapeutic strategy for targeting miRNA-mediated pathways in metastatic disease.

DELLA proteins, members of the GRAS-domain family of transcriptional regulators, are critical for plant growth and development. They modulate transcription indirectly via interactions with hundreds of transcription factors. The phytohormone gibberellin (GA) triggers DELLA degradation, providing a mechanism by which plants can integrate developmental and environmental signals to regulate gene expression and optimize growth responses. In agriculture, DELLA mutations have been instrumental in improving crop performance. Most modern wheat ( Triticum aestivum L.) varieties carry Rht-B1b or Rht-D1b alleles that encode DELLA proteins resistant to GA-mediated degradation, resulting in constitutive partial suppression of stem growth, a semi-dwarf stature and lodging resistance. However, these alleles also reduce nitrogen use efficiency and early vigour, limiting their utility in some environments. Understanding how DELLA proteins regulate growth and development is, therefore, critical for refining breeding strategies. In this study, we identified the orthologous C2H2 zinc-finger transcription factors INDETERMINATE DOMAIN 5 ( IDD5 ) in wheat and SEMI-DWARF 3 ( SDW3 ) in barley ( Hordeum vulgare ) as positive regulators of stem and leaf expansion. Both IDD5 and SDW3 physically interact with, and act downstream of, DELLA proteins as key components of GA-mediated growth responses. Altered expression levels of GA biosynthesis genes suggest that IDD5 helps maintain GA homeostasis in addition to growth regulation. Loss-of-function mutations in IDD5 and SDW3 confer a GA-insensitive semi-dwarf phenotype comparable to that of the Rht-D1b ‘Green Revolution’ allele, highlighting their potential as novel dwarfing alleles for cereal improvement. Significance statement Our study identifies homologous wheat and barley transcription factors (IDD5 and SDW3) that interact with DELLA proteins to regulate plant height. Unlike conventional ‘Green Revolution’ DELLA mutations, which can reduce height but also have drawbacks such as lower nitrogen-use efficiency, these IDD genes may provide more targeted approaches to manage plant growth. By showing how IDD proteins promote stem and leaf expansion and by revealing their potential as alternative dwarfing alleles, our research opens new avenues both for fundamental research into plant growth pathways and for applications in cereal breeding. Ultimately, it could help produce crops with improved lodging resistance and fewer negative side effects than current dwarfing alleles.

The phospholipid scramblases Xkr8 and TMEM16F externalize phosphatidylserine (PS) by distinct mechanisms. Xkr8, is activated by caspase-mediated proteolytic cleavage, and in synergy with inactivation of P4-ATPase flippases, results in the irreversible externalization of PS on apoptotic cells and an “eat-me” signal for efferocytosis. In contrast, TMEM16F is a calcium activated scramblase that reversibly externalizes PS on viable cells via the transient increase in intracellular calcium in live cells. The tumor microenvironment (TME) is abundant with exposed PS, resulting from prolonged oncogenic and metabolic stresses and high apoptotic indexes of tumors. Such chronic PS externalization in the TME has been linked to host immune evasion from interactions of PS with inhibitory PS receptors such as TAM and TIM receptors. Here, in an effort to better understand the contributions of apoptotic vs live cell PS-externalization to tumorigenesis and immune evasion, we employed an E0771 orthotopic breast cancer model and genetically ablated Xkr8 and TMEM16F using CRISPR/Cas9. While neither the knockout of Xkr8 nor TMEM16F showed defects in cell intrinsic properties related to proliferation, tumor-sphere formation, and growth factor signaling, both knockouts suppressed tumorigenicity in immune-competent mice, but not in NOD/SCID or RAG-KO immune-deficient strains. Mechanistically, Xkr8-KO tumors suppressed macrophage-mediated efferocytosis, and TMEM16F-KO suppressed ER stress/calcium-induced PS externalization. Our data support an emerging idea in immune-oncology that constitutive PS externalization, mediated by scramblase dysregulation on tumor cells, supports immune evasion in the tumor microenvironment. This links apoptosis/efferocytosis and oncogenic stress involving calcium dysregulation, contributing to PS-mediated immune escape and cancer progression.

Extrachromosomal DNA (ecDNA) is a common source of oncogene amplification across many types of cancer. The non-Mendelian inheritance of ecDNA contributes to heterogeneous tumour genomes that rapidly evolve to resist treatment. Here, using single-cell and live-cell imaging, single-micronucleus sequencing, and computational modelling, we demonstrate that elevated levels of ecDNA predisposes cells to micronucleation. Damage on ecDNA, commonly arising from replication stress, detaches ecDNA from the chromosomes upon which they hitchhike during cell division, thereby causing micronucleus formation in daughter cells. Clusters of oncogene-containing, CIP2A-TOPBP1-associated ecDNA molecules form, and asymmetrically segregate into daughter cell micronuclei during cell division. ecDNA chromatin remains highly active during mitosis, but upon micronucleation, it undergoes suppressive chromatin remodeling, largely ceasing oncogene transcription. These studies provide insight into the fate of damaged ecDNA during cell division.

Summary The function of HSFs, known otherwise as ‘master thermoregulators,’ in plant developmental remains largely uninvestigated. In this study, we strategically analyze SlHSFB3a , a class B sub member of HSF transcription factor family, uniquely expresses in age-dependent tomato roots and improves root architecture by synchronizing auxin homeostasis. This data demonstrates SlHSFB3a overexpressed transgenics display higher lateral root (LR) density and early LR emergence improving root architecture. Generation of CRISPR-Knockout mutants displayed contrasting phenotype, confirming SlHSFB3a ’s vital role in root growth. In SlHSFB3a manipulated roots, concentration gradient auxin responses oscillated with increase in LR number. We highlight the signal transduction of SlHSFB3a mediated auxin activation that enhances tomato LRs. SlHSFB3a directly inhibits auxin repressors, increases auxin flow via ARF7/LOB20 pathway and positively modulates LR growth.

Abstract Intracellular parasites like Toxoplasma gondii scavenge host nutrients, particularly lipids, to support their growth and survival. Although Toxoplasma is known to adjust its metabolism based on nutrient availability, the mechanisms that mediate lipid sensing and metabolic adaptation remain poorly understood. Here, we performed a genome-wide CRISPR screen under lipid-rich (10% Fetal Bovine Serum (FBS)) and lipid-limited (1% FBS) conditions to identify genes critical for lipid-responsive fitness. We identified the Toxoplasma protein GRA38 as a lipid-dependent regulator of parasite fitness. GRA38 exhibits phosphatidic acid (PA) phosphatase (PAP) activity in vitro, which is significantly reduced by mutation of its conserved DxDxT/V catalytic motif. Disruption of GRA38 led to the accumulation of PA species and widespread alterations in lipid composition, consistent with impaired PAP activity. These lipid imbalances correlated with reduced parasite virulence in mice. Our findings identify GRA38 as a metabolic regulator important for maintaining lipid homeostasis and pathogenesis in Toxoplasma gondii. 

Abstract Human papillomavirus (HPV) infection is a major threat to women’s health worldwide. High-risk subtypes, particularly HPV16, require rigorous screening and long-term surveillance to control cervical cancer. However, traditional HPV testing is hampered by the need for nucleic acid extraction, reliance on specialized technicians, and fluorescence detection equipment, limiting its suitability for rapid on-site testing. In this study, we developed a Concanavalin A-assisted extraction-free one-pot recombinase polymerase amplification (RPA) CRISPR/Cas12a assay (ConRCA) for HPV16. Concanavalin A-coated magnetic beads were used for target enrichment and nucleic-acid-extraction-free processing. Suboptimal protospacer-adjacent motifs were used to achieve a one-pot RPA–CRISPR/Cas12a assay. The ConRCA assay can be completed in approximately 25 min under isothermal conditions and can detect at least 1.2 copies/µL of HPV16 genomic DNA using a fluorescence reader or test strip. The feasibility of this detection method was evaluated with 31 unextracted clinical samples. Compared with qPCR, the overall sensitivity was 95% (19/20), and the specificity was 100% (11/11). Our results indicate that the ConRCA assay has great potential utility as a point-of-care testing for the rapid identification of HPV.

Abstract Mild cognitive impairment (MCI) is an intermediate stage between normal cognition and dementia, with a high risk of progression to Alzheimer’s disease (AD). As an important threshold for the intervention and prevention of AD, accurate diagnosis of AD-related MCI based on plasma biomarkers is crucial. However, early detection of AD-related MCI still poses a huge challenge. Herein, we propose an ultrasensitive CRISPR-based multi-protein detection array (UCMDA) that integrates the array device with CRISPR/Cas12a and antibody pair-based multi-recombinase polymerase amplification to facilitate inexpensive, sensitive, and specific detection of multiple markers in plasma. With only one fluorescence probe, UCMDA can simultaneously detect Aβ40, Aβ42, p-tau181, p-tau217, p-tau231, and p-tau396,404 within 1 h with a detection limit of 1 fg/mL. The applicability of UCMDA was demonstrated in 155 clinical plasma samples from normal senile controls (NCs), individuals with AD-related MCI, and patients with AD. We found that the UCMDA platform combined with machine learning algorithms achieved accurate early diagnosis of AD-related MCI with a detection accuracy, sensitivity, and specificity exceeding 90.38%. We anticipate that this inexpensive, ultrasensitive, and multi-detection platform can also be widely used to detect other protein-related biomarkers besides AD.

While prime editing offers improved precision compared to traditional CRISPR-Cas9 systems, concerns remain regarding potential off-target effects, including epigenetic changes such as DNA methylation. In this study, we investigated whether prime editing induces aberrant CpG methylation patterns. Whole-genome bisulfite sequencing revealed overall methylation similarity between Cas9-edited, and PE2-edited cells. However, localized epigenetic changes were observed, particularly in CpG islands and exon regions. The PE2-edited group showed a higher proportion of differentially methylated regions (DMRs) in some coding sequences compared to controls and Cas9-edited samples. Notably, CpG island methylation reached 0.18% in the PE2 vs. Cas9 comparison, indicating a higher susceptibility of these regulatory elements to epigenetic alterations by prime editing. Gene ontology and KEGG pathway analyses further revealed enrichment in molecular functions related to transcriptional regulation and redox activity in PE2-edited cells. These findings suggest that prime editing, while precise, may introduce subtle but functionally relevant methylation changes that could influence gene expression and cellular pathways. In summary, prime editing can induce localized DNA methylation changes in human cells, particularly within regulatory and coding regions. Understanding these epigenetic consequences is critical for the development of safer and more effective therapeutic applications of genome editing technologies.

Sirex noctilio is an invasive pest of pine that has caused significant economic damage in South Africa and many other Southern Hemisphere countries. Current management tools are not efficient in all cases and consequently there is a need for more efficient and targeted control measures. An emerging tool for pest management is the use of gene editing and associated gene drive systems. In this study, we aim to investigate the use of CRISPR-Cas gene drive systems in the management of S. noctilio in South Africa. As a first step, we developed a model for the population dynamics of S. noctilio , using historical national population monitoring data and incorporating the influence of two main biological control agents of the pest. We then modelled the influence of two different CRISPR-Cas systems on the population dynamics of S. noctilio namely, a baseline CRISPR model and Complementary Sex Determination CRISPR (CSD) model. Each model is used to simulate a male and female only introduction strategy to estimate the effectiveness of different methods of introducing the gene drive system. The model calibration was achieved by optimizing the model fit to existing data using the least squares technique. Results suggest that both CRISPR gene drive systems would be effective at controlling the population growth of S. noctilio at high levels of introduction, but overall population control would be hindered by practical limitations. Although only two CRISPR models were explored, the underlying population model serves as a framework for further studies into the population dynamics of Sirex noctilio , as well as many other CRISPR-Cas gene drive systems.

ABSTRACT Objectives The defense mechanisms in bacterial pathogens protect them from host immune systems, bacteriophage infections and stringent environmental conditions. This study explores the defense-systems in multidrug resistant Klebsiella pneumoniae isolated from Ghanaian hospital ICUs focusing on CRISPR-cas, restriction-modification and toxin-antitoxin systems (TAs). Method Genomic DNA of K. pneumoniae environmental (NS2) and clinical (PS4) strains were subjected to whole genome sequencing using Illumina and assembled with SPAdes (v3.13.1). CRISPR-cas, restriction-modification and TAs were identified using PADLOC, defense finder and TADB3.0 respectively. Results The strains harbor diverse defense systems. Relative to reference K. pneumoniae with 10 defense systems, NS2 has twelve and PS4, five. CRISPR-Cas systems were found only in NS2, while both strains have type I, II and IV restriction and modification systems. The strains have > 30 characterized and novel TAs (type I, II, IV, VIII) similar to reference K. pneumoniae . NS2 harbors more TAs than PS4 both on chromosomes and plasmids. The strains have comparable resistance determinants to more than six classes of antibiotics. Conclusion The genome of strains encodes similar clinically relevant defense-systems indicating possibility of microbial exchange from fomites and humans. They could leverage the defense-systems to propagate resistance in high-risk environments such as the hospital. Fomite-resident strain with high levels of resistance could increase infection risk in the ICU; hence, should also be prioritized.

Some RNA viruses package their genomes with extraordinary selectivity, assembling protein capsids around their own viral RNA while excluding nearly all host RNA. How the assembling proteins distinguish viral RNA from host RNA is not fully understood, but RNA structure is thought to play a key role. To test this idea, we perform in-cellulo packaging experiments using bacteriophage MS2 coat proteins and a variety of RNA molecules in E. coli . In each experiment, plasmid-derived RNA molecules with a specified sequence compete against the cellular transcriptome for packaging by plasmid-derived coat proteins. Following this competition, we quantify the total amount and relative composition of the packaged RNA using electron microscopy, interferometric scattering microscopy, and high-throughput sequencing. By systematically varying the input RNA sequence and measuring changes in packaging outcomes, we are able to directly test competing models of selective packaging. Our results rule out a longstanding model in which selective packaging requires the well-known TR stem-loop, and instead support more recent models in which selectivity emerges from the collective interactions of multiple coat proteins and multiple stem-loops distributed across the RNA molecule. These findings establish a framework for understanding selective packaging in a range of natural viruses and virus-like particles, and lay the groundwork for engineering synthetic systems that package specific RNA cargoes. Significance Statement Bacteriophage MS2 packages its RNA genome into protective protein shells called capsids while excluding nearly all host-cell RNA. Engineering synthetic capsids with similar selectivity could enable a broad range of RNA-based technologies, including CRISPR gene editing systems, mRNA vaccines, and other emerging RNA-based therapeutics. Our study shows that selective packaging in MS2 is not dictated by a single, high-affinity RNA-protein interaction but instead emerges from the collective interactions of multiple coat proteins and an ensemble of stem-loops distributed across the RNA molecule. By establishing these collective interactions as the basis of selectivity, our findings provide a foundation for engineering synthetic capsids capable of selectively packaging target RNAs for next-generation RNA-based technologies.

Long noncoding RNAs (lncRNAs), non-protein-coding transcripts exceeding 200 nucleotides, are critical regulators of gene expression through chromatin remodeling, transcriptional modulation, and post-transcriptional modifications. While ionizing radiation (IR) induces cellular damage through direct DNA breaks, reactive oxygen species (ROS)-mediated oxidative stress, and bystander effects, the functional involvement of lncRNAs in radiation response remains incompletely characterized. Here, through genome-wide CRISPR activation (CRISPRa) screening in non-small cell lung cancer (NSCLC) cells, we identified LOC401312 as a novel radiosensitizing lncRNA, the stable overexpression of which significantly enhanced IR sensitivity. Transcriptomic profiling revealed that LOC401312 transcriptionally upregulates carbamoyl-phosphate synthase 1 (CPS1), a mitochondrial enzyme involved in pyrimidine biosynthesis. Notably, CPS1 overexpression recapitulated the radiosensitization phenotype observed with LOC401312 activation. Mechanistic investigations revealed that CPS1 suppresses the phosphorylation of ATM kinase (Ser1981) and XRCC1 protein levels, which are key mediators of DNA damage checkpoint activation and base excision repair, respectively. This study establishes the LOC401312-CPS1-ATM/XRCC1 axis as a previously unrecognized regulatory network governing radiation sensitivity, highlighting the potential of lncRNA-directed metabolic rewiring to impair DNA repair fidelity. Our findings not only expand the functional landscape of lncRNAs in DNA damage response but also provide a therapeutic rationale for targeting the LOC401312-CPS1 axis to improve radiotherapy efficacy in NSCLC.

The primate brain possesses unique physiological and developmental features whose systematic investigation is hampered by a paucity of transgenic germline models and tools. Here, we present a minimally invasive method to introduce transgenes widely across the primate cerebral cortex using ultrasound-guided fetal intracerebroventricular viral injections (FIVI). This technique enables rapid-onset and long-lasting transgene expression following the delivery of recombinant adeno-associated viruses (rAAVs). By adjusting the gestational timing of injections, viral serotypes, and transcriptional regulatory elements, rAAV FIVI allows for systematic targeting of specific cell populations. We demonstrate the versatility of this method through restricted laminar expression in the cortex, Cre-dependent targeting of neurons, CRISPR-based gene editing, and labeling of peripheral somatosensory and retinal pathways. By mimicking key desirable features of germline transgenic models, this efficient and targeted method for gene transfer into the fetal primate brain opens new avenues for experimental and translational neuroscience across the lifespan.

Abstract Epithelial ovarian cancer (EOC) metastasizes predominantly through multicellular aggregates known as spheroids, which disseminate within the peritoneal cavity and initiate secondary disease upon reattachment at distant sites. EOC spheroids resist detachment-induced cell death by upregulating stress responses including AMP-activated protein kinase (AMPK) signaling and AMPK-dependent macroautophagy (autophagy), highlighting these pathways as potential therapeutic targets. Previously, we used a pharmacological approach to putatively identify Ca 2+ /calmodulin-dependent protein kinase kinase 2 (CAMKKβ, encoded by CAMKK2 ) as the primary activator of AMPK in EOC spheroids. Herein we have generated CAMKK2 knockout EOC cell lines via CRISPR–Cas9 genome editing to confirm this function of CAMKKβ and explore the impacts of its loss using in vitro and in vivo models of metastatic EOC. CAMKK2 knockout spheroids exhibited decreased AMPK activation, autophagic flux, cell viability, and metastatic potential relative to parental spheroids, and intraperitoneal xenograft tumours lacking CAMKKβ grew slower than their CAMKKβ-intact counterparts. Effect magnitudes varied between cell line models, suggesting context-dependent roles for CAMKKβ in EOC and rationalizing further studies to characterize the underlying mechanisms. Altogether, our findings highlight CAMKKβ as an important contributor to metabolic reprogramming in EOC spheroids and as a potential therapeutic target in the setting of advanced disease.

Genome editing has the potential to treat genetic disorders at the source. This can be achieved by modifying the defective DNA through the intentional insertion, deletion, or substitution of genomic content. Among all genome editing technologies, CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) is considered the gold standard. CRISPR/Cas uses a single guide RNA (sgRNA) to direct the Cas nuclease to a target DNA region. Due to the ease at creating small RNA molecules, it is possible to have the CRISPR/Cas complex target any arbitrary DNA sequence, thus making it a versatile tool. The efficacy of the complex is dependent on the ability of the sgRNA to bind to a complementary DNA sequence, which varies based on the sequence. Thus, a major challenge is finding sgRNA sequences that have good efficacy. This is where computational models can aid scientists: by predicting the activity of sgRNAs to help narrow the search space of finding the optimal sgRNA. We have used a large new dataset to build and compare the ability of several different machine learning architectures’ ability to predict on-target CRISPR/Cas activity. Additionally, we explored how adding GC content affects our sgRNA activity predictions. Our novel hybrid model, ChromeCRISPR, combines the strengths of Convolutional Neural Networks (CNN) with Recurrent Neural Network (RNN) models, has outperformed state-of-the-art models, including DeepHF and AttCrispr, establishing a new benchmark for predictive accuracy in CRISPR/Cas9 efficacy predictions.