Bacillus methanolicus represents a thermophilic methylotroph whose methanol utilization depends on plasmid-encoded genes. It serves as a unique model for deciphering plasmid-dependent methylotrophy and an ideal chassis for low-carbon biomanufacturing using CO2-derived C1 substrates. Despite its evolutionary uniqueness and industrial potential, the lack of synthetic biology tools has hindered both mechanistic understanding and strain engineering. Here, we present a comprehensive synthetic biology platform comprising a high-efficiency electroporation protocol, a CRISPR method enabling robust and multiplex genome editing, diverse neutral loci for gene integration and overexpression, and a cloud-based genome-scale metabolic model iBM822 for user-friendly biodesign. Leveraging this toolkit, we systematically dissected plasmid-dependent methylotrophy, host restriction-modification systems, and functional significance of the chromosomal methylotrophic genes through targeted deletion. To address plasmid loss-induced strain degeneration, we integrated the large endogenous plasmid pBM19 into the chromosome for stable and intact methylotrophic growth. Finally, by integrating metabolic modeling with CRISPR editing, we engineered L-arginine feedback regulation to achieve the first L-arginine biosynthesis from methanol. This study establishes a synthetic biology framework for B. methanolicus, promoting mechanistic exploration of methylotrophy and low-carbon biomanufacturing.

As well as undergoing mutational selection, bacterial genomes are shaped by a complex evolutionary interplay among diverse accessory genome elements (AGEs). In this study we define AGEs as encompassing both autonomous elements including phage, plasmids, integrative conjugative elements (ICEs) and carried elements such as defence systems (DSs) and antimicrobial resistance genes (ARGs). Genomic studies can facilitate our better understanding of the relationships among these AGEs within microbial genomes, offering deeper insights into their roles in bacterial adaptation and evolution. Emerging evidence suggests that bacterial DSs can interact both synergistically and antagonistically with each other, and ecological studies describe non-random patterns of co-occurrence and avoidance of DSs in bacterial populations. Here, we analysed the distributions of DSs and other AGEs in a recently curated dataset of 4,288 Pseudomonas aeruginosa genomes. Genomic DS content varied by ecological niche, with higher numbers per genome in non-CF isolates (average n=7.9) compared to CF isolates (average n=6.9). We observed multiple phylogenetically independent associations (n=426) and dissociations (n=50) among DSs, and among other AGEs, many of which had a plausible biological basis. We further explored the relative importance of interactions among different AGEs, revealing that DSs and anti-defence systems engage in the most significant interactions with other AGEs and, most notably, with each other. Ultimately, these patterns of DS interactions and their variation across ecological niches reveals the evolutionary conflicts shaping bacterial accessory genomes and provides a significant public resource for downstream studies on DS and other AGE interactions.

Patients who are recipients of allogeneic transplants or have underlying autoimmune disease require immune suppression, often with calcineurin inhibitors (CNI). There is an expanding repertoire of immune effector cell (IEC) therapies, including CD19 CAR-T cells and viral-specific T cells (VSTs), deployed in these patients; however, ongoing CNI therapy may be detrimental to IEC function. We thus developed a CRISPR/Cas9-based approach to engineer dual CNI [cyclosporine (CsA) and voclosporin (VCS)] resistant IEC therapies by targeting PPIA (encoding cyclophilin A - CypA), a critical binding partner for both drugs. Because CypA has several homeostatic functions in T cells, a complete CypA knock-out could impair cell viability. To avoid this, we edited the last exon of the PPIA gene, corresponding to the C-terminus of CypA, selectively disrupting amino acids that mediate CsA/VCS-based inhibition, while leaving the majority of CypA intact. Unlike an edit in an upstream exon, which was detrimental to cell survival and rapidly selected out, C-terminal editing was stable throughout expansion and preserved CypA protein expression. This edit was then introduced into two types of IECs. Edited CD19 CAR-T cells retained in vitro effector function in the presence of CsA/VCS, including preserved proliferation, target cell killing, and cytokine production. Edited CMV-specific T cells demonstrated antigen-specific proliferation and cytokine production in the presence of CsA/VCS. This report of site-specific CypA modification offers a promising avenue for developing next-generation IECs that should function effectively in patients receiving CsA/VCS and thus expand applications for adoptive cell therapies in multiple clinical settings. Key Points CRISPR editing of the last exon of PPIA retains CypA expression but with an altered C-terminus that disrupts CsA and VCS interactions PPIA Δ C immune effector cells demonstrate retained proliferation and function in the presence of CsA and VCS

Trichoderma atroviride is a well-known mycoparasitic fungus widely used for the biological control of fungal plant pathogens. Expanding its genetic toolbox is essential to facilitate efficient genetic manipulation, including successive transformations and multiple or reusable selection markers for consecutive gene deletions. We applied CRISPR/Cas9 via ribonucleoprotein (RNP) complexes for gene editing in T. atroviride and successfully deleted three target genes, i.e., pks4 (involved in spore pigment production), pyr4 (pyrimidine biosynthesis), and pex5 (receptor for peroxisomal matrix protein import). Although double-strand breaks induced by Cas9 can be repaired via homology-directed repair (HDR), using donor templates, the most effective gene deletions in our case were achieved via non-homologous end joining (NHEJ), by co-transforming a transiently stable telomere vector carrying the hygromycin-resistance gene ( hph ), which was rapidly lost under non-selective conditions. This strategy promoted NHEJ repair and resulted in the efficient deletion of open reading frames between two Cas9 target sites. Our results demonstrate that combining CRISPR/Cas9 RNP delivery with transient telomere vectors provides a fast and reliable method for marker-free gene deletion and vector recycling in T. atroviride , advancing the reverse genetic toolkit available for this important biocontrol fungus.

Abstract Background: Thermus thermophilus HB27 is a promising thermophilic chassis for recombinant thermostable protein production, owing to its high optimal growth temperature, which can simplify downstream processing and reduce contamination risks. However, maximizing its potential requires optimized genetic tools and host strains. Key limitations include a shortage of well-characterized strong constitutive promoters and potential degradation of recombinant proteins by proteases. To address these, we established a β-galactosidase reporter system (endogenous TTP0042) to screen for strong constitutive promoters and investigated the impact of deleting specific protease genes on protein expression. Results: Screening of 13 endogenous promoter regions identified P0984 as exhibiting significantly 13-fold higher activity than the control promoter driving the reporter gene. Constructing a plasmid-free strain (HB27ΔpTT27) successfully minimized 270 kb of the genome; it exhibited auxotrophy for cobalamin (requiring 0.1 μg/ml AdoCbl for growth) and a slightly reduced growth rate compared to the wild-type, while its transformation efficiency remained comparable. Notably, a CRISPR-deficient precursor strain (HB27ΔIII-ABΔI-CΔ CRF3 ) showed a significant (~100-fold) increase in transformation efficiency compared to the wild-type, facilitating subsequent genetic manipulations. Systematic knockout of 16 predicted non-essential protease loci was performed. Characterization revealed that deletion of TTC0264 (putative ClpY/HslU) and TTC1905 (putative HhoB) significantly reduced extracellular proteolytic activity. Iterative deletion based on phenotypic analysis led to strain DSP9 (10 protease loci deletions), which maintained robust growth and exhibited enhanced accumulation of the β-galactosidase reporter protein compared to the parental strains. Conclusions: This study provides foundational advancements for T. thermophilus HB27 chassis development, and genetic tools represent valuable resources for optimizing T. thermophilus as a platform for heterologous thermostable protein production and ideas for antibiotic-free systems.

Cdc42 is a Rho-family GTPase conserved across eukaryotes, where it plays essential roles in cell polarization. In single-celled yeast systems, Cdc42 is a key driver of symmetry breaking and polarized growth, forming zones of activity that locally recruit eRectors to organize the cytoskeleton and polarize secretion. Here we show that Cdc42 also functions in cell-cell fusion during Schizosaccharomyces pombe sexual reproduction but concentrates at the fusion site through mechanisms distinct from those proposed in Saccharomyces cerevisiae . Notably, the cdc42-mCherry SW allele (but not the cdc42-sfGFP SW allele), which is functional for cell polarization and has been used across organisms for dynamic studies, exhibits a strong fusion defect. These cells block fusion before cell wall digestion but after actin fusion focus formation, indicating that Cdc42 is required to translate the vesicle cluster into polarized cargo delivery. We trace the defect to instability of Cdc42-mCherry SW and demonstrate that cell fusion requires higher Cdc42 protein levels than mitotic polarized growth. Remarkably, by constructing an allelic series driving Cdc42 expression over a 5-fold range, we discover that polarized growth responds linearly to Cdc42 protein levels, whereas sexual reproduction exhibits a sharp switch-like response. Thus, the topology of the Cdc42 regulatory network is distinct for its polarization and mating functions.

The subcellular positioning of organelles is critical to their function and is dynamically adapted to changes in cell morphology. Yet, how cells sense shifts in their dimensions and redistribute organelles accordingly remains unclear. Here we reveal that cell-size-scaling of mitochondria distribution and function is directed by polarised trafficking of mRNAs. We identify a 29bp 3’UTR motif in mRNA encoding TRAK2, a key determinant of mitochondria retrograde transport, that promotes cell-size-dependent targeting of TRAK2 mRNA to distal sites of cell protrusions. Cell-size-scaled mRNA polarisation in turn scales mitochondria distribution by defining the precise site of TRAK2-MIRO1 retrograde transport complex assembly. Consequently, 3’UTR motif excision perturbs size-regulated transport and eradicates scaling of mitochondria positioning, triggering distal accumulation of mitochondria and progressive hypermotility as cells increase size. Together, our results reveal an RNA-driven mechanistic basis for the cell-size-scaling of organelle distribution and function that is critical to homeostatic control of motile cell behaviour.

Photosynthesis and respiration are fundamental metabolic processes in plants, tightly connected through shared substrates, energy dynamics, and redox balance. Arabidopsis is the key genetic model for plants but monitoring these sorts of physiological processes presents significant challenges using traditional gas-exchange or fluorescence-based techniques due to the small size of intact Arabidopsis thaliana (arabidopsis) seedlings. Here, we validate and characterize the use of Clark-type oxygen electrodes, specifically the Hansatech Oxytherm+P system, to quantify both photosynthetic and respiratory activity in intact arabidopsis seedlings. By monitoring oxygen evolution in dark and light phases, we demonstrate that oxygen consumption and production correspond to mitochondrial respiration and photosynthesis, respectively. These processes were modulated by tissue biomass, light intensity, developmental stage, and stress conditions. Specific inhibitors such as potassium cyanide and paraquat confirmed that the recorded changes in oxygen concentrations reflected mitochondrial cytochrome oxidase activity and photosystem electron transport-dependent oxygen production, respectively. Moreover, oxygen evolution increased significantly with bicarbonate supplementation, validating the system's sensitivity to carbon fixation. We further showed that photosynthetic activity measured with this method correlates with a quantitative green index and responds dynamically to de-etiolation, abiotic stress (salt, osmotic, oxidative), and temperature shifts. Our study lays the groundwork for measuring photosynthesis based on oxygen evolution and respiration in arabidopsis knockout mutants, CRISPR lines, overexpression lines and ecotypes using Clark-type oxygen electrodes and highlights key considerations and limitations to consider when applying this approach. This platform could also be adapted for many other small tissue plant samples.

In Drosophila melanogaster, bag of marbles ( bam ) encodes a protein essential for germline stem cell daughter (GSC) differentiation in early gametogenesis. Despite its essential role in D. melanogaster , direct functional evaluation of bam in other closely related Drosophila species reveal this essential function is not necessarily conserved. In D. teissieri , for example, bam is not essential for GSC daughter differentiation. Here, we generated bam null alleles using CRISPR-Cas9 in a species more distantly related to D. melanogaster, D. americana , to interrogate whether bam ’s essential GSC differentiation function is novel to the melanogaster species group or a function more basal to the Drosophila genus. To further characterize the extent of the functional flexibility of other GSC regulating genes, we generated a gene ortholog dataset for 366 GSC regulating genes essential in D. melanogaster across 15 additional Drosophila and two outgroup species. We find that bam ’s essential GSC function is conserved between D. melanogaster and D. americana and therefore originated prior to the formation of the melanogaster species group. Additionally, we find that ∼8% of the 366 GSC genes essential in D. melanogaster are absent in at least one of the 17 species in our ortholog dataset. These results indicate that developmental systems drift (DSD), in which the specific genes regulating a function may change, but the final phenotype is retained, occurs in stem cell regulation and the production of gametes across Drosophila species. Article summary Results: from CRISPR induced bam null mutants in D. americana and comparative ortholog analysis of essential GSC regulating genes indicate that the evolutionary origin of bam ’s essential GSC differentiation function is likely basal to the Drosophila genus, and there is functional flexibility in at least ∼8% of the 366 GSC regulating genes across the 17 included species.

Macrophages in the tumor microenvironment exert potent anti-tumorigenic activity through phagocytosis. Yet therapeutics that enhance macrophage phagocytosis have not improved outcomes in clinical trials for patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). To systematically identify regulators of phagocytosis, we performed genome-scale CRISPR knockout screens in human leukemia cells co-cultured with human monocyte-derived macrophages. Surprisingly, we found that whereas the classic “don’t eat me” signal CD47 inhibited mouse macrophages, it did not inhibit phagocytosis by human macrophages. In contrast, the O-linked glycosylation and sialylation pathways were strong negative regulators of phagocytosis. In AML, the cell surface O-linked glycoprotein CD43 was the major effector of the O-linked glycosylation and sialylation pathways. Genetic deletion or antibody blockade of CD43 enhanced macrophage phagocytosis. This work highlights the importance of using human platforms to identify immune checkpoints, and nominates CD43 as a glyco-immune regulator of human macrophage phagocytosis.

Most genetic variants associated with human traits and diseases lie in noncoding regions of the genome 1 , and a key challenge is determining which genes they affect 2,3 . A common approach has been to leverage associations between natural genetic variation and gene expression to identify expression quantitative trait loci (eQTLs) in the population 4,5 . At the same time, a newer method uses pooled CRISPR interference (CRISPRi) perturbations of noncoding loci with single-cell transcriptome sequencing 6,7 . Here, we systematically compared the results from these approaches across hundreds of genomic regions associated with blood cell traits. We find that while the two approaches often identify the same target genes, they also capture distinct features of gene regulation. CRISPRi tends to detect genes that are physically closer to regulatory variants and more constrained, whereas eQTL studies are sensitive to detecting multiple, often distal, genes. By comparing these discoveries to a gold-standard set of genes linked to blood traits, we demonstrate that the two approaches provide highly complementary insights with distinct strengths and caveats. Our results offer guidance for improved design of CRISPRi and eQTL studies and highlight their potential as a powerful toolkit for interpreting disease-associated loci.

Human congenital anomalies account for twice the mortality of childhood cancer. Despite advancements in genome sequencing and transgenic mouse models that have aided in understanding their pathogenesis, significant gaps remain. Through a forward genetics approach, we previously discovered the hypo-morphic anteater allele of Cse1l which displayed variable craniofacial phenotypes. To circumvent the variability seen in this model, we generated a conditional allele of Cse1l and genetically ablated it in the dorsal midline giving rise to portions of the nervous system and the cranial neural crest cells using the Wnt1-Cre 2 driver. Our analysis revealed that Wnt1-Cre2; Cse1l CRISPR/flox embryos exhibited severe malformations in the forebrain, midbrain, and hindbrain, accompanied by a dramatic hypoplasia of the frontonasal, maxillary, and mandibular processes, and the second pharyngeal arch. Wnt1-Cre2; Cse1l CRISPR/flox embryos were embryonic lethal by E11.5 likely due to defects in the ventricular myocardium. Wnt1-Cre2; Cse1l CRISPR/flox embryos exhibited consistently increased apoptosis at E9.5 in the affected tissues along with an increase in p53 expression. These data together show a previously unknown critical function of CSE1L in neural crest cell survival during development. Summary Statement Cse1l is critical for neural crest cell survival and genetic ablation of Cse1l in neural crest cells resulted in dramatic apoptosis with increase in p53 expression.

Abstract Heifer Infertility and disease are important challenges in dairy cattle production. Here, genetic differences between Holstein heifers with varying fertility potential and health were investigated. A genome-wide association analysis was carried out to compare heifers that conceived at first insemination against those requiring multiple attempts or failing to become pregnant, as well as heifers culled due to health issues. There were 12 significant SNPs (P<5x10 -5 ) associated with fertility and 35 SNPs associated with health traits. There were 166 significant SNPs when infertile, sub-fertile and animals culled due to health issues were grouped. Two SNPs identified in the analysis of infertility were found near NUFIP1 and within TENM4 genes, both genes are linked to embryonic lethality in mouse knockouts. Follow-up CRISPR-Cas9-mediated disruption of NUFIP1 significantly (P<0.05) reduced in vitro blastocyst development in cattle embryos, while TENM4 editing did not alter in vitro blastocyst development. Additionally, SNPs overlapped with previously identified reproduction-related QTL ( CNTN4 , DLG2 , PARP10 , PRICKLE , TMEM150B ) or health-related QTL ( FAM162A , PARP10 ). There were genes within or near genes previously associated with age at menarche ( CADM2, DLG2 , FHIT , LSAMP and TENM4 ) or lung function or pulmonary diseases ( ASCC2 , BCAS3 , BTBD9 , CADM2 , CNTN4 , CPEB4 , CTNNA2 , DEUP1 , DGKH , DLG2 , ENOX1 , EPHB1 , ERC2 , ERGIC1 , EYA2 , FAM162A , FGF18 , FHIT , GRID1, KCNIP4 , LINGO2 , LRMDA , MALRD1 , NEBL , PLA2G6 , PLXDC2 , PRPF18 , SLC8A1 , TEAD4 , TSPAN9 ) in humans. These results further support genetic components of fertility and health in cattle. The findings also show overlapping genetic architecture between heifer fertility and health traits, with a degree of conservation across mammals.

High-throughput genomic studies have uncovered associations between diverse genetic alterations and disease phenotypes; however, elucidating how perturbations in functionally disparate genes give rise to convergent cellular states remains challenging. Here, we present PerturbFate, a high-throughput, cost-effective, combinatorial-indexing single-cell platform that enables systematic interrogation of massively parallel CRISPR perturbations across the full spectrum of gene regulation, from chromatin remodeling and nascent transcription to steady-state transcriptomic phenotypes. Using PerturbFate, we profiled over 300,000 cultured melanoma cells to characterize multi-modal phenotypic and gene regulatory responses to perturbations in more than 140 Vemurafenib resistance-associated genes. We uncovered a shared dedifferentiated cell state marked by convergent transcription factor (TF) activity signatures across diverse genetic perturbations. Combined inhibition of cooperative TF hubs effectively reversed cellular adaptation to Vemurafenib treatment. We further dissected phenotypic responses to perturbations in Mediator Complex components, linking module-specific biochemical properties to convergent gene activations. Together, we reveal common regulatory nodes that drive similar phenotypic outcomes across distinct genetic perturbations. We also delineate how perturbations in functionally unrelated genes reshape cell state. PerturbFate thus establishes a versatile platform for identifying key molecular regulators by anchoring multi-modal regulatory dynamics to disease-relevant phenotypes.

Objective Catastrophic antiphospholipid syndrome (CAPS), characterized by widespread thrombosis and multi-organ failure, is associated with high morbidity and mortality. We previously established complement activation as a pathogenic driver of CAPS and identified rare germline variants in complement-regulatory genes including Complement Receptor 1 ( CR1 ) in 50% of CAPS. Methods We quantified CR1 expression by flow cytometry across hematopoietic cell types. CRISPR/Cas9 genome editing of TF-1 (erythroleukemia) cells was performed to generate CR1 “knock-out” and “knock-in” lines with patient-specific CR1 variants. Multiomics analysis was performed to investigate the role of methylation in CR1 expression in patients with reduced CR1 expression. Functional impact of low CR1 expression was assessed by complement-mediated cell killing using modified Ham (mHam) assay, cell-bound complement degradation products through flow cytometry and circulatory immune complexes (CIC) in serum samples through ELISA. Results CR1 expression in erythrocytes was markedly reduced on CAPS erythrocytes (n=9, 21.80%) compared to healthy controls (HC; n=32, 82.40%), with promoter hypermethylation emerging as a plausible epigenetic mechanism for CR1 downregulation. A novel germline variant ( CR1- V2125L; rs202148801 ) mitigated CR1 expression and increased complement-mediated cell death of knock-in cell lines. Erythrocytes from the patient with the CR1- V2125L variant had low CR1 expression. Levels of CIC, which are bound and cleared by CR1 on erythrocytes, were higher in acute CAPS (n=3, 25.55 µg Eq/ml) than healthy controls (n=3, 7.445 µg Eq/ml). Five patients were treated with C5 inhibition which mitigated thrombosis. Conclusion Genetic or epigenetic-mediated CR1 deficiency is a potential hallmark of CAPS and predicts response to C5 inhibition.

mRNA translation involves multiple regulatory steps, but how translation elongation in-fluences protein output remains unclear. Using SunTag live-cell imaging and mathematical modeling, we quantified translation dynamics in single mRNAs across diverse coding sequences. Our Totally Asymmetric Exclusion Process (TASEP)-based model revealed a strong coordination between initiation and elongation rates, resulting in consistently low ribosome density (≤12% occupancy) across all reporters. This coupling persisted under pharmacological inhibition of the elongation factor eIF5A, where proportional decreases in both initiation and elongation rates maintained homeostatic ribosome density. In contrast, eIF5A knockout cells exhibited a significant decrease in ribosome density, suggesting altered coordination. Together, these results highlight a dynamical coupling of initiation and elongation rates at the single-mRNA level, preventing ribosome crowding and maintaining translational homeostasis in mammalian cells.

Atrial fibrillation (AF) is a common arrhythmia with a complex genetic basis, yet the molecular mechanisms linking rare and common variants remain unclear. Using induced pluripotent stem cell-derived atrial cardiomyocytes, we uncover a novel mechanism by which a rare pathogenic LMNA variant encoding Lamin A/C disrupts chromatin accessibility and gene regulation at AF-associated loci. Specifically, reduced accessibility at an SCN5A enhancer harboring an AF-associated variant leads to reduced sodium current, conduction abnormalities, and re-entrant AF. These electrophysiological defects are rescued by CRISPR-mediated activation of the SCN5A promoter and enhancer, providing the first molecular evidence of epistatic gene-gene interactions driving arrhythmia risk and mechanistically linking atrial myopathy and AF. At the population level, we demonstrate that carriers of LMNA protein-altering variants with a high polygenic risk score are at a two-fold increased risk of early-onset AF, highlighting the need to integrate rare and common variants for more accurate AF risk assessment.

ABSTRACT Amyotrophic lateral sclerosis (ALS) caused by mutation in superoxide dismutase 1 ( SOD1 ) accounts for 15-30% of familial ALS and is typically autosomal dominant. How single base pair/amino acid changes in this small protein cause neurodegeneration is unknown. In North America, SOD1 A4V is the most common familial ALS SOD1 mutation and results in an aggressive form of ALS. Here, we present a novel genomically humanised mouse model of SOD1 A4V , in which the mouse Sod1 locus has been replaced by the human SOD1 gene, with intact genomic architecture of exons and introns, but bearing an A4V mutation. In agreement with previously reported human genomic knock-in mice, the phenotype is mild; however, transcriptomic and metabolomic profiling reveal significant dysregulation of glycolysis, the tricarboxylic acid (TCA) cycle, and lipid metabolism. These changes suggest an early bioenergetic imbalance that precedes neuromuscular impairment. Our findings support metabolic dysfunction as an early event in ALS pathogenesis. This freely available SOD1 A4V model provides a valuable tool for studying ALS progression and identifying therapeutic targets for pre-symptomatic treatment. SUMMARY STATEMENT This study describes the generation and analysis of novel genomically humanised SOD1 A4V mice, revealing metabolic dysfunction through integrated multi-omic analyses, characterising a freely available potential pre-symptomatic ALS model for future research.

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) occurs as a complex, multifaceted event driven by the interplay of tumor permissive genetic mutations, nature of cellular origin and microenvironmental stress. In this study, using primary human pancreatic acinar 3D organoids, we performed CRISPR knockout screen targeting 199 previously underappreciated potential tumor suppressors curated from clinical PDAC samples. Our data revealed significant enrichment of a list of candidates, with NF2 emerging as the top target. Functional validation confirmed that loss of NF2 promotes the transition of PDAC to an invasive state, potentially through extracellular matrix modulation. NF2 inactivation was found to enhance PDAC cell fitness under nutrient starvation. This adaptation not only reinforces the oncogenic state but also confers therapeutical resistance. Additionally, we found that NF2 loss is associated with the fibroblast heterogeneity and cancer-stroma communications in tumor evolution. These findings establish NF2 as a critical tumor suppressor in PDAC and uncover its role in mediating nutrient adaptation and drug resistance. Importantly, this study provides new insights into drug resistance mechanisms and potential therapeutic targets in PDAC.

The modification of mosquito populations at scale through CRISPR-Cas9-mediated homing gene drives is a promising route for malaria vector control. Integral gene drives (IGDs) are designed to utilize the regulatory sequences of endogenous genes to express only the minimal set of components required for gene drive. In this study, we describe the creation and characterization of the nanos d IGD targeting and inserted into the nanos gene of the malaria vector Anopheles gambiae and show that it achieves high rates of gene drive (97.7% in females, 99.0% in males). We find that homozygous nanos d females but not males show impaired fecundity, and a variable loss of ovary phenotype. Transcriptomic analysis of ovaries points to decreased transcript levels of the nanos gene when harbouring Cas9. As a minimal genetic modification, nanos d does not induce widespread transcriptomic perturbations, and its susceptibility to Plasmodium spp. and O’nyong nyong virus infection remains similar to wild-type mosquitoes. Importantly, we find that nanos d propagates efficiently in caged mosquito populations and is maintained as a source of Cas9 after the emergence of drive resistant alleles whilst also mobilising a non-autonomous antiparasitic effector modification. The nanos d gene drive shows promise as a genetic tool for malaria vector control via population modification, and we outline steps towards its further optimization.

CRISPR/Cas12a, recognized for its high efficiency and specificity in nucleic acid recognition, has found extensive applications in infectious disease sensing. Nonetheless, a significant challenge persists in devising a simple method for the rapid, convenient, and accurate visualization of detection results. In this study, we demonstrate an innovative and simple CRISPR-on-beads assay for visual detection of methicillin-resistant Staphylococcus aureus (MRSA). This approach combines isothermal target amplification, CRISPR/LbCas12a-mediated cleavage, and magnetic bead-based probe binding for signal amplification, enabling rapid and convenient identification of bacterial DNA. Our results show that this detection method exhibits both high sensitivity and specificity, with a detection limit as low as 30 CFU/μL. This newly developed biosensing approach can easily be integrated with a portable fluorescence microscope for medical diagnostics in resource-limited settings.

ABSTRACT Trypanosoma brucei , the causal agent of Human and Animal African trypanosomiasis proliferates in the extracellular milieu of mammals. It acquires host macromolecular nutrients, by receptor mediated endocytosis. The best characterised receptor is for transferrin (TfR) and it has been reported to be sequestered in the flagellar pocket, the sole site of endocytosis. In this location the TfR may be inaccessible to adaptive immune system effectors. The T. brucei genome encodes ∼15 TfR variants, and here we compared two, the first attached to the plasma membrane by a single glycosylphosphoinositol (GPI)-anchor and the other by two. Transferrin uptake kinetics were similar and rapid for both. Unexpectedly, initial binding of transferrin occurred over the whole cell surface suggesting the TfR was not sequestered in the flagellar pocket. This localisation was confirmed by immunofluorescence assays and was independent of the number of GPI-anchors. Two other GPI-anchored receptors were investigated to determine whether localisation to the whole cell surface was a general property of GPI-anchored receptors. Haptoglobin-haemoglobin uptake assays and immunofluorescence localisation of complement factor H receptor showed both were also whole cell surface localised. The mechanisms by which trypanosome receptors are protected from antibody-mediated attack are more subtle than hiding in a pocket.

Abstract Cytokinin signal transduction occurs through a "two-component system." Type-A and -C response regulators (RRs) are groups of proteins of similar structures constituting significant components of cytokinin signal transduction. In rice, 13 (Type-A) and two (Type–C) RRs have been identified to date; however, their functions remain partially known. In this study, we examined the expression patterns of Type-A and Type-C RRs in rice using RNA-Seq and confirmed their functions by constructing mutants of the 15 genes with CRISPR/Cas9. Almost all Type-A RRs played positive roles in the development of secondary branches and secondary spikelets, particularly RR2 and RR4 . Notably, rr1 rr2 and rr8 rr12 rr13 higher-order mutants displayed small panicle sizes and decreased plant height. Additionally, both Type-C RRs played positive roles in regulating heading date. RNA-seq revealed several genes with significantly altered expression in the rr2 and rr4 mutants, with almost half of the differentially expressed genes (DEGs) overlapping between the two mutants. Many of the DEGs were associated with the cytokinin and abscisic acid pathways.Our findings provide new insights into the functions of Type-A and -C RRs in rice growth and may serve as a foundation for future studies focusing on cytokinin signaling.

Abstract Background Cultivated tomato is susceptible to necrotrophic pathogens Botrytis cinerea and Alternaria solani . No dominant resistance against these pathogens has been reported in wild relatives of tomato. Results Through screening of a tomato Micro-Tom EMS population we identified a mutant that showed decreased susceptibility to both necrotrophic fungi. Previously, we reported a mutation in the tomato PUB17 gene as the cause of reduced susceptibility in this mutant. Surprisingly, M4 progeny of one M3 plant homozygous for the pub17 mutation showed segregation with some plants displaying an even higher level of resistance than the pub17 mutant. This highly resistant progeny was shown to contain a mutation in tomato PUB21 in addition to the mutation in PUB17 . The role of PUB21 as a susceptibility factor for both necrotrophic fungi was confirmed in RNAi-silenced and CRISPR-mutated transformants. Conclusions In this study we identified a new PUB gene, SlPUB21 , involved in susceptibility of tomato to necrotrophic pathogens. We showed that mutation of this gene resulted in increased resistance against these pathogens.

Abstract Archaeal antiviral defense systems remain poorly characterized despite recent advances in understanding prokaryotic immunity. Here, we analyze 7,747 archaeal genomes, the largest and most diverse dataset to date, revealing a striking disparity in defense system prevalence and diversity compared to Bacteria. Nearly one-third of archaeal genomes lack known systems beyond CRISPR-Cas and restriction-modification (in contrast to only 2.2% bacterial genomes), and only 31% contain CRISPR-Cas systems, far below previous estimates. While many known defense systems appear restricted to Bacteria, several single-gene candidate systems (PDCs) are enriched in Archaea. Phylogenetic analyses suggest that PDC-S27, PDC-S70, and PDC-M05 likely originated in Archaea, representing rare archaeal contributions to the prokaryotic immune repertoire. Consistent with earlier studies, our findings support the existence of deep evolutionary links between archaeal and eukaryotic systems for argonautes and viperins. These analyses highlight both the underexplored nature and the evolutionary significance of archaeal immunity, calling for expanded efforts to uncover archaeal-specific systems and improve our understanding of immune evolution across domains of life.