Abstract Staphylococcus aureus Cas9 (SaCas9) is smaller than the widely used Streptococcus pyogenes Cas9 (SpCas9) and has been harnessed for gene therapy using an adeno-associated virus vector. However, SaCas9 requires a longer NNGRRT (where N is any nucleotide and R is A or G) protospacer adjacent motif (PAM) for target DNA recognition, thereby restricting the targeting range. Furthermore, the precise nuclease activation mechanism of SaCas9 remains elusive. Here, we rationally engineered a SaCas9 variant (eSaCas9-NNG) with an expanded target scope and reduced off-target activity. The eSaCas9-NNG induced indels and base conversions at endogenous sites bearing NNG PAMs in human cells and mice. We further determined the cryo-electron microscopy structures of eSaCas9-NNG in five distinct functional states, revealing the structural basis for the improved specificity and illuminating notable differences in the activation mechanisms between the small SaCas9 and the larger SpCas9. Overall, our findings demonstrate that eSaCas9-NNG could be used as a versatile genome editing tool for in vivo gene therapy, and improve our mechanistic understanding of the diverse CRISPR-Cas9 nucleases.

Efficient purification remains one of the major bottlenecks in the development of plant-based systems for recombinant protein production. The complex metabolites, particularly polyphenols, which usually cause recombinant protein aggregation during purification. In this study, we identified two key polyphenol oxidase genes, PPOa and PPOb from N.benthamiana as responsible for these effects. Using CRISPR/Cas9, we generated two ppoa;ppob double knockout lines that significantly improved the purification of functional proteins like SARS-CoV-2 Spike trimer and influenza HA trimer. These lines showed reduced polyphenol-protein interactions, minimized aggregation, and higher purification yields. Our work establishes a clean, high-efficiency N. benthamiana chassis for scalable recombinant protein production.

ABSTRACT The mitochondrial disulphide relay is the key machinery for import and oxidative protein folding in the mitochondrial intermembrane space. Among IMS proteins with unknown function, we identified FAM136A as a new substrate of the mitochondrial disulphide relay. We demonstrate a transient interaction between FAM136A and MIA40, and that MIA40 introduces four disulphide bonds in two twin-CX 3 C motifs of FAM136A. Consequently, IMS import of FAM136A requires these cysteines and its steady state levels in intact cells are strongly dependent on MIA40 and AIFM1 levels. Furthermore, we show that FAM136A forms non-covalent homodimers as a mature protein. Acute deletion of FAM136A curtails cellular proliferation capacity and elicits a robust induction of the integrated stress response, coincident with the aggregation and/or depletion of selected IMS proteins including HAX1 and CLPB. Together, this establishes FAM136A as a pivotal component of the IMS proteostasis network, with implications for overall cellular function and health.

Mutations in the ATRX chromatin remodeller predispose to a developmental genetic disorder and cancer, but how it safeguards genome and telomere stability remains unresolved. Here, we uncover critical dependencies for the CTC1-STN1-TEN1 (CST) complex and RAD9A-HUS1-RAD1 (9-1-1) clamp in ATRX deficient cells. ATRX:CST synthetic lethality manifests following accumulation of telomeric G-rich ssDNA, which results in telomere loss and cell death. Conversely, we attribute ATRX:9-1-1 synthetic lethality to genome-wide ssDNA lesions, which compromise DNA replication. We further show ATRX suppresses DNA damage during replication stress by counteracting the activity of the FAM111A protease. We demonstrate that roles of ATRX in telomere maintenance and replication are genetically separable requiring its ATPase activity and PIP-box, respectively, and independently of its DAXX interaction. Collectively, functions of ATRX in suppressing toxic ssDNA lesions are context-dependent and are key to global DNA replication and telomere integrity.

Skeletal muscle formation involves the fusion of myocytes into precisely aligned, multinucleated myofibres. These fibres continue to grow through reiterative rounds of myocyte fusion, incorporating new myonuclei and supporting muscle growth, repair and regeneration over organismal life span. The vertebrate-specific myocyte fusogens, Myomaker (Mymk) and Myomixer (Mymx), are crucial for generating multinucleated skeletal muscles. While the role of the transmembrane protein Mymk is well established, expression dynamics of mymx and the function of the Mymx micropeptide is less well understood. Here, using quantitative imaging and a mymx knockout strain, we explored the impact on myogenesis at different life stages of the zebrafish. We demonstrate that during the initial phase of muscle formation, mymx has a spatiotemporally varied expression across all axes of the developing myotome. On Mymx loss, myotome morphogenesis is disrupted, with both cell and tissue structure impacted. Moreover, we could show differential effects of Mymk versus Mymx loss on myocyte fusion and muscle growth. Finally, we report that perturbation to adult muscle multinucleation and size impacted bone development, again with different phenotypic severities among the two fusogen mutants. Together, our work provides key insights into the interplay between myocyte fusion, myotome morphogenesis and acquisition of final adult form.

Parkinson’s disease (PD) is characterized by α-synuclein accumulation and dopaminergic neuron degeneration, with dopamine (DA) oxidation emerging as a key pathological driver. However, the mechanisms underlying this neurotoxic process remain unclear. Using PD patient-derived and CRISPR-engineered iPSC midbrain dopaminergic neurons lacking DJ-1, we identified defective sequestration of cytosolic DA into synaptic vesicles, which culminated in DA oxidation and α-synuclein accumulation. In-depth proteomics, state-of-the-art imaging, and ultrasensitive DA probes uncovered that decreased VMAT2 protein and function impaired vesicular DA uptake, resulting in reduced vesicle availability and abnormal vesicle morphology. Furthermore, VMAT2 activity and vesicle endocytosis are processes dependent on ATP, which is notably reduced in DJ-1-deficient dopaminergic neurons. ATP supplementation restored vesicular function and alleviated DA-related pathologies in mutant dopaminergic neurons. This study reveals an ATP-sensitive mechanism that regulates DA homeostasis through VMAT2 and vesicle dynamics in midbrain dopaminergic neurons, highlighting enhanced DA sequestration as a promising therapeutic strategy for PD. Teaser Loss of DJ-1 interferes with VMAT2 function and vesicle dynamics, leading to DA oxidation and α-synuclein pathology in PD neurons.

Summary Opioids are potent analgesics often prescribed for the treatment of chronic pain, a condition affecting millions worldwide. Although pain states increase vulnerability to opioid use disorders, the neural mechanisms underlying this interaction remain incompletely understood. The ventral tegmental area (VTA) is a key site for opioid actions, and emerging evidence suggests that pain states and opioid experience both induce transcriptional, molecular, and circuit adaptations in the VTA that contribute to motivated behaviors. However, the transcriptional responses of distinct VTA cell types to each of these factors (alone or in combination) have not been identified. Here, we employed single-nucleus RNA sequencing to comprehensively define transcriptional alterations in the rat VTA to acute morphine administration in a chronic inflammatory pain model. We report that morphine induces gene expression changes primarily in glial cells and dopamine neurons, with minimal effects in other neuronal cell types. Surprisingly, VTA astrocytes and oligodendrocytes exhibited the most robust transcriptional responses to opioid exposure, despite lacking detectable opioid receptor expression. Among the most highly regulated glial genes was Fkbp5 , which encodes a co-chaperone protein that acts in concert with heat shock proteins to modulate stress responses. Using pharmacological and CRISPR-based approaches in rat glial cells and human astrocytes, we demonstrate that regulation of Fkbp5 is mediated indirectly through glucocorticoid signaling rather than direct opioid receptor activation. These findings reveal that glial cells within reward circuits undergo profound transcriptional reprogramming in response to opioids through indirect, stress-hormone mediated mechanisms, highlighting a previously unappreciated non-neuronal contribution to opioid-induced neural adaptations.

The clinical deployment of antibiotics is undermined by antimicrobial resistance. Without new agents to treat antibiotic resistant bacterial infections, mortality rates are predicted to reach 10 million people per year by 2050. Most antibiotics are derived from natural products (NPs) produced by bacteria; however, this resource was abandoned by industry because of high rediscovery rates. We are amid a natural product renaissance fuelled by inexpensive access to genome sequencing and sophisticated bioinformatic tools, which have highlighted that most of the biosynthetic pathways for NPs are not expressed in the laboratory. Here, we engineered the expression of a silent biosynthetic gene cluster harboured by an environmental isolate of Streptomyces albidoflavus . By using a bioinformatics-guided approach, we isolated and structurally characterised a novel glycopeptide antibiotic (GPA) named biffamycin A, which is the smallest GPA known and harbours unprecedented 5-chloro-4-methoxy tryptophan and 3-hydroxy(α- d -mannoysl)- d -lysine moieties. Biffamycin A possesses antimycobacterial and antistaphylococcal bioactivity, including methicillin-vancomycin-resistant Staphylococcus aureus .

Replication and segregation of the nucleus and kinetoplast, the mitochondrial DNA, are tightly coordinated in trypanosomatid parasites, but the signalling pathways that govern this process are unknown. Here, we characterise the mitotic spindle kinase (MSK), a key regulator of this coordination in Leishmania . Using chemical genetics, we engineered an analog-sensitive MSK to inhibit its activity. We show that inhibition of MSK impairs mitotic spindle elongation and blocks both nuclear and kinetoplast segregation, halting cell cycle progression and leading to cell death. We combined chemical genetics with proximity-based phosphoproteomics to identify four substrates: two GTPase-activating proteins, a nuclear segregation protein, and a hypothetical protein. We demonstrate that MSK co-localises with these four proteins in the nucleus, mitotic spindle, kinetoplast, and cytoplasm. Our findings establish MSK as a critical kinase that controls the co-ordinated segregation of the nucleus and kinetoplast, providing a new avenue for understanding cell cycle regulation in Leishmania .

Maintenance of genome integrity is essential for cellular homeostasis, and its perturbation leads to tumorigenesis. Here, we uncover an unanticipated somatic role for the synaptonemal complex protein SYCP1—previously regarded as strictly meiosis-specific—in a broad spectrum of human cancers including breast cancer. Through integrative genomic, proteomic, and functional analyses, we demonstrate that SYCP1 is aberrantly re-expressed in tumor cells, where it actively promotes DNA damage repair, cell cycle progression, and malignant growth. SYCP1 binds chromatin at regulatory elements and directly controls transcriptional programs governing genome maintenance, including key effectors such as CCNB1 , PCNA , RAD51C , and H2AX . Loss of SYCP1 impairs DNA repair kinetics, attenuates tumor cell proliferation and migration, and increases sensitivity to chemotherapeutics cisplatin and gemcitabine. Mechanistically, SYCP1 interfaces with chromatin remodeling complexes and transcription factors SP1 and SP2, modulating their genomic occupancy and facilitating oncogenic transcriptional outputs. Clinically, high SYCP1 expression stratifies patients with poor prognosis and therapy resistance across multiple cancer types. Our findings illuminate a previously unrecognized moonlighting function of SYCP1 in somatic cancer cells and position it as a critical chromatin-associated regulator of genome stability, with implications for biomarker development and therapeutic targeting.

ABSTRACT Background Bcl-2-associated athanogene 3 (BAG3) is a mediator of chaperone assisted selective autophagy, and in the brain, most highly expressed in astrocytes. However, its role in astrocytes remains poorly defined. Given the genetic and pathological links of BAG3 to proteostasis and neurodegenerative diseases, we investigated how BAG3 contributes to astrocyte function and Alzheimer’s disease (AD). Methods SnRNA-seq of the human brain determined cell type expression of BAG3. CRISPR/Cas9 gene editing in human iPSCs, followed by tandem mass tag-mass spectrometry and RNA-sequencing was performed to assess proteomic and transcriptomic changes following BAG3 loss. Co-immunoprecipitation of BAG3 in human astrocytes defined the interactome, with top interactors being validated by western blot (WB), AlphaFold modeling, and proximity ligation assays. In astrocytes, autophagic flux, lysosomal phenotypes, proteasome activity, and endocytic uptake were measured in BAG3 KO and BAG3 WT. Finally, BAG3 expression was assessed in postmortem AD brain by WB and snRNA-seq, and its functional relevance to amyloid-β (Aβ) degradation was tested in co-cultures of BAG3 KO iAs with familial AD neurons. Results In human brain and iPSC models, BAG3 was most highly expressed in astrocytes. Further, BAG3 loss caused greater proteomic disruption in astrocytes than in neurons. In the absence of BAG3, astrocytes showed reduced autophagy, diminished lysosome abundance and activity, and decreased proteasome function. To uncover molecular binding partners of BAG3 that might influence these phenotypes, we performed co-immunoprecipitation, revealing interactions with HSPB8 and other heat shock proteins, proteasome regulators (PSMD5, PSMF1), and the retromer component, VPS35. Integration of BAG3 KO transcriptomic and proteomic datasets pinpointed AD-relevant proteins under post-translational control of BAG3, which included GFAP, BIN1, and HSPB8. HSPB8 levels were markedly reduced in BAG3-deficient astrocytes with overexpression partially rescuing its levels. Loss of astrocytic BAG3 impaired Aβ clearance in co-culture with APP/PSEN1 mutant neurons, directly linking BAG3 to a disease-relevant astrocyte function. Finally, analysis of postmortem brain tissue revealed BAG3 marks a stress-responsive astrocyte subtype in the brain of aged individuals with AD. Conclusions BAG3 binds to key regulators of autophagy, proteasome activity, and retromer function to coordinate astrocyte proteostasis, lysosomal function, and Aβ clearance. These findings position BAG3 as a potential therapeutic target and coordinator of glial protein quality control in neurodegeneration.

CRISPR-based gene drive can address ecological problems by biasing their inheritance coupled with an effector for either population modification of suppression. However, the risk of uncontrolled spread impedes some applications of gene drive. Daisy chain drives have received much attention as a potential approach to overcome this problem. They potentially allow efficient spread in a target population but are ultimately self-limiting. This is achieved by splitting a standard gene drive into multiple dependent elements, where each element can bias the inheritance of another, except one non-driving element. With the successive loss of each chain link, spread of transgenic elements will slow down and eventually stop. Here, we use modelling to assess the population dynamics of suppression daisy chain drives in both panmictic and continuous space models. We find that achieving population elimination through a single release of daisy chain gene drives is possible but difficult, with relatively high requirements for drive performance and release size. These effects are substantially amplified in spatial models. We also constructed two configurations of daisy chain gene drives in Drosophila melanogaster as a proof-of-principle. One is a rescue drive for population modification, and the other aims for population suppression by targeting a female fertility gene. Both functioned within expectations at moderate efficiency in individual crosses. However, the system failed to spread in cage populations because of higher than expected fitness costs. Overall, our study demonstrates that daisy chain systems may be promising candidates for both modification and suppression, but challenges remain in both construction and potential deployment in large regions.

ABSTRACT Genome-wide association studies have identified >1,000 loci associated with clinically important red blood cell (RBC) traits, such as hemoglobin concentration and cell volume. However, few of these associations have been characterized at the molecular level such that most causal genes and variants remain elusive. Here, we performed pooled CRISPR screens in an erythroid cell line to identify genes and regulatory non-coding sequences that control RBC density. We perturbed 556 candidate genes and genomic sequences near 2,114 GWAS variants. We used a density gradient to detect the impact of these CRISPR perturbations on cell density. After validation, we found 17 genes and 13 regions near GWAS variants that regulate cell density. Some of these genes have previously been implicated in RBC biology (e.g. ATP2B4 , CCND3 , EPOR ) although many are novel (e.g. CHTF8 , CTU2 , DNASE2 ). We confirmed that deletions in the osmotic stress response kinase gene OXSR1 increase cell density, and a phosphoproteome analysis in OXSR1-depleted cells indicated that this phenotype is accompanied with a dephosphorylation of the upstream kinase WNK1 and the downstream target KCC3 ( SLC12A6 ). We also combined CRISPR perturbations and RNA-sequencing to show how a non-coding genomic sequence near rs13255015 regulates the expression of the transcription factor ZFAT in cis and SLC4A1 in trans . SLC4A1 encodes Band3, a known regulator of RBC hydration and volume. Our results suggest experimental strategies to characterize GWAS findings and provide new molecular insights into the regulation of complex RBC traits.

Extracellular vesicles (EVs) are lipid-delineated nanoparticles that are produced by most cell types. EVs contain complex molecular cargoes that can have useful therapeutic or vaccine immunological effects. Cell-free gene expression systems can be used to produce membrane proteins in vitro , that can co-localise and integrate with exogenously added EVs. To advance this type of cell-free EV engineering we manufactured, isolated and characterised HEK293 cell EVs. These EVs were successfully cell-free engineered with several CD63-based membrane fusion proteins. In our most optimal conditions, up to 4.83 ×10 11 /ml of HEK293 EVs were successfully cell-free engineered with a fusion membrane protein incorporating CD63 I-shaped membrane-insertion topology transmembrane helix 3 (CD63ITM3) and monomeric green lantern (mGL). Finally, we also demonstrated that nano flow cytometry is a powerful tool for assessing cell-free EV engineering efficiency. In the future cell-free EV engineering could help accelerate future EV discoveries and the development of EV translational applications. Graphical abstract Technology readiness Cell-free systems are a well-established part of the engineering biology toolkit. Indeed, several applications are already at TRL9 stage, including commercially available cell-free protein synthesis kits, drug screening assays, field-tested medical biosensors, as well as therapeutic antibody manufacturing that has now reached cell-free reaction volume scales of up to 4500 L. However, cell-free extracellular vesicle (EV) engineering is currently at TRL3 / 4 stage. Whilst our study helps to further advance cell-free EV engineering, key challenges remain. To accelerate technology readiness, improvements in scalable EV isolation methods, increased eukaryotic cell-free protein synthesis yields, and assay automation will likely speed up cell-free EV engineering workflows. Furthermore, artificial intelligence-guided design workflows could be used to rapidly create community accessible libraries of EV membrane protein scaffolds, therapeutic, cell targeting and other modular elements for use in EV engineering studies. Finally, the development of EV potency assays for functional assessment of cell-free engineered EVs are needed to progress prototyped designs towards clinical translation. If these challenges are addressed, we envision that cell-free EV engineering will become an important approach in EV biological research and clinical applications. Highlights Cell-free extracellular vesicle (EV) engineering could be utilised to rapidly prototype and test novel biotechnological, vaccine, or therapeutic EVs for foundational or translational applications. Our data highlight some of the impacts that hollow fibre-based cell culture, EV isolation methods and different cell-free engineering approaches have on cell-free EV engineering workflows. We demonstrate several characterisation assays and technologies, including nanoflow cytometry, that can be used to assess cell-free EV engineering efficiency.

The recent advent of long-read whole genome sequencing has enabled us to create an accurate telomere-to-telomere reference genome, construct pangenome graphs, and compile precise catalogs of genomic structural variations (SVs). These comprehensive SV repositories provide an excellent opportunity to explore the role of SVs in genotype-phenotype associations and examine the mechanisms by which SVs are introduced through double-strand break (DSB) repair. Here, we employed comprehensive SV catalogs identified through various short- and long-read whole genome sequencing efforts to infer the underlying mechanisms of SV introduction based on their genomic and epigenomic profiles. Our findings indicate that high local DNA methylation and DNA shape-related features, such as low variations in propeller twist, support the origins of homology-driven SVs. Subsequently, we utilized an active-learning-based unsupervised clustering approach, revealing that the homology-dependent SVs show greater evidence of retaining ancestral recombination patterns compared to their homology-independent counterparts. Finally, our comparison of inherited and de novo SVs from healthy populations and rare disease cohorts showed distinct upstream H3K27me3 levels in de novo SVs from individuals with ultra-rare disorders. These findings highlight genome-wide characteristics that may influence the choice of repair mechanisms linked to heritable SV origins.

RNA is at the forefront of therapeutics and gene editing technologies. Yet, RNA synthesis remains expensive and low-yield. Consequently, most oligo manufacturers abstain from synthesizing RNA oligos longer than 60-mers. Solid-phase synthesis is the current standard production method but is often fraught with low coupling yields for canonical nucleotides and even poorer coupling for modifications. This results in high levels of byproducts such as truncations and RNA infidelity. Existing analytical methods can only provide quality control metrics such as RNA length distribution or limited composition information for short oligos. Here, we developed a standard quality control metric using Oxford Nanopore direct RNA sequencing to obtain direct insight into RNA length distribution, sequence, and presence of RNA modification sites. Our pipeline identifies error-prone regions and truncation sites that occur during synthesis. Furthermore, problematic steps in the synthesis are identified and repaired. We show that our platform can produce and assess CRISPR guide RNAs with high-fidelity and higher cleavage activity, and further, that modifications can be reliably detected. We envision that our tool will serve as an integral method for quality control pipelines that assess the integrity and accuracy of synthetic RNAs and guide the improved synthesis and yield of synthesized RNAs. Graphical Abstract

Prochlorococcus is the most numerically abundant photosynthetic organism in the oceans and plays a role in global carbon cycling. Despite its ecological significance and the availability of over a thousand assembled genomes, progress in understanding gene function has been limited by the lack of genetic tools. Here, we report a reproducible electroporation-based protocol to introduce replicative plasmids into two strains of Prochlorococcus representing different ecotypes: MIT9313 (low-light adapted) and MED4 (high-light adapted). Using plasmids carrying a spectinomycin resistance cassette, we achieved transformation in ~33% of MED4 and ~10% of MIT9313 attempts, with greatest success when electroporating cells in late exponential phase. Transformed cells stably retained plasmids and expressed resistance genes, demonstrating functional uptake and gene expression. We also delivered a modified 13 kb plasmid carrying a CRISPR-Cpf1 system into MED4. While no targeted edits were observed, cpf1 and specR were expressed, indicating successful delivery of large constructs and active transcription. These findings represent a key step toward genetic manipulation of Prochlorococcus , enabling future optimization of gene editing approaches and deeper functional analysis of its vast and largely uncharacterized pangenome.

Background Climate warming promotes the expansion of insect pests. Among the inducible defense responses activated by attacked plants, Kunitz trypsin protease inhibitors (KTIs) play an outstanding role. KTIs affect food digestion and thereby control the fitness of herbivorous insects. Poplars contain an expanded family of KTIs, whose distinct intrinsic functions are under investigation. Here, we set out to identify KTIs with anti-herbivore activity and assessed the potential growth trade-off incurred by high KTI expression levels. Results We identified 28 KTIs in the haploid genome of Populus x canescens , 21 of them were responsive to herbivory. The greatest induction was observed for KTI_400, KTI_600 and KTI_0882 ( P. trichocarpa orthologues Potri.019G124400, Potri.019G124600, Potri.019G088200), whereas a moderate response was found for KTI_53200 (Potri017G153200 orthologue), a protein mainly localized in the xylem sap. Mechanical wounding and methyl-jasmonate resulted in fast and strong induction of KTI_400 and KTI_600 and moderate or lacking responses in KTI_0882 and KTI_53200 . Increased KTI expression levels were associated with upregulation of ALLENE OXIDE SYNTHASE (AOS), whereas exposure to compounds eliciting ethylene or salicylic acid signaling did not affect KTI s. We generated stable CRISPR/Cas12a-mediated knock-out and p35S -mediated overexpression lines of KTI_400, KTI_600 and KTI_53200 in Populus x canescens. Among the wildtype and transgenic lines, only kti_400+kti_600 double knock-out lines produced greater biomass. Larvae of Helicoverpa armigera, a pest expanding in Europe due to a warmer climate, were allowed to feed on wildtype and transgenic poplar lines. Transgenic poplars overexpressing KTI_400 or KTI_600 resulted in reduced and their double knock-out lines in increased weight gain of the larvae. In contrast, overexpressing or knock-out lines of KTI_53200 had no effect on larval weight gain compared with controls. Conclusion KTI_400 and KTI_600 are potent, natural in-planta anti-herbivorous agents. Their expression is associated with larval growth reductions. Modulation of KTI_53200 levels had no direct effects on the fitness of leaf-feeding H. armigera or on plant growth. This study sheds light on the potential application of KTI in plant defenses and biocontrol against herbivores in trees and presents new options to investigate growth-defense theories.

Heterochromatin Protein 1α (HP1α) is a fundamental component of constitutive heterochromatin, forming subnuclear condensates whose regulation and function remain poorly understood. Here, we present an image-based CRISPR screen targeting nuclear factors that identifies splicing as a pivotal pathway regulating HP1α condensates. We discovered that unspliced intronic RNA modulates HP1α condensates by interacting co-transcriptionally with HP1α. By modulating the intron content, RNA processing restricts HP1α-RNA interactions at chromatin, thus enabling heterochromatin organization. Disruption of HP1α condensates due to enhanced interactions with unspliced RNA leads to loss of heterochromatin and the activation of stress response protective genes. We propose that RNA is a central component of heterochromatin that modulates HP1α condensates, and that RNA processing enzymes act as a surveillance mechanism for condensates by dynamically regulating the network of multi-valent interactions between RNA and chromatin factors. This model underscores the crosstalk between chromatin organization, transcription, and RNA processing, potentially governing broader nuclear functions.

Phosphoinositide-specific phospholipase C (PI-PLC) is a signaling enzyme that hydrolyzes membrane phosphoinositide to generate lipid- and lipid-derived second messengers. In plants, PI-PLCs have been implicated in various physiological processes, including immunity. Tomato SlPLC2 was previously shown to be implicated in susceptibility to the necrotrophic fungus Botrytis cinerea . However, whether this observation extends to pathogens with different lifestyles and evolutionary origins, remains unknown. Here, we investigated SlPLC2 function during infection with Phytophthora infestans , a globally devastating oomycete and causal agent of potato and tomato late blight. CRISPR-Cas9 knockout SlPLC2 plants showed significantly reduced disease symptoms, lower pathogen biomass, and decreased sporangia production, indicating impaired colonization. Upon infection, SlPLC2 knockout lines displayed attenuated expression of salicylic acid (SA)- and jasmonic acid (JA)-responsive genes suggesting disrupted hormone signaling. Consistent with PLC role during plant defense, early immune responses such as hydrogen peroxide accumulation and callose deposition were reduced in knockout plants. At the cellular level, these plants allow fewer infection vesicles formation by P. infestans , accompanied by an increase in expression of a biotrophy-associated effector gene PiAvrblb2 , suggesting impaired establishment of infection. In Nicotiana benthamiana SlPLC2-GFP localizes predominantly at the plasma membrane. Upon P. infestans inoculations, SlPLC2-GFP localizes to the membrane surrounding infection vesicles, where we also detected phosphoinositides PI4P and PI(4,5)P₂. Overexpression of SlPLC2 in Nicotiana benthamiana enhanced susceptibility to P. infestans , reinforcing its positive role in colonization. Together, these findings identify SlPLC2 as a susceptibility factor associated with enhanced P. infestans colonization. Statement This study identifies SlPLC2 as a key susceptibility factor that supports Phytophthora infestans infection by coordinating vesicle formation and early immune responses in tomato.

Extrachromosomal DNA (ecDNA) is a major driver of oncogene amplification, intratumoral heterogeneity and therapy resistance across multiple cancer types, yet there are currently no effective strategies to selectively eliminate it. Here, we show that the type I-E CRISPR system can specifically target and degrade ecDNA in human cancer cell lines (COLO320DM and GBM39). By designing guide RNAs targeting ecDNA-specific breakpoints absent from chromosomal DNA, we achieved efficient depletion of MYC- and EGFR-containing ecDNAs in COLO320DM and GBM39 cells. Loss of ecDNA was accompanied by diminished oncogene signaling, disrupted ecDNA architecture, and impaired tumor cell proliferation, without detectable chromosomal off-target activity. These findings establish a proof-of-concept framework for directly targeting oncogenic ecDNAs and highlight type I-E CRISPR as a promising platform for therapeutic development in ecDNA-driven cancers.

ABSTRACT Cystic fibrosis (CF) is a devastating genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator ( CFTR ) gene. As morbidity and mortality from CF results from a lack of mucus clearance that leads to chronic bacterial infections and progressive loss of lung function, site-specific insertion of a CFTR cDNA into the endogenous CFTR locus in airway basal stem cells (ABSCs) could prove curative for all disease-causing mutations. This study describes the development of nonviral genome editing reagents, designed to be packaged into nonviral delivery systems. An sgRNA targeting the 5’ untranslated region of CFTR was characterized as directing high on-target cutting and displaying a safe off-target profile. Airway cell lines electroporated with chemically-modified (1-Aminohexane - AmC6), linear double-stranded DNA (ldsDNA) constructs were utilized as an Homology Directed Repair (HDR) donor, initially optimized with an mCitrine reporter. Expectedly, when the 780bp mCitrine cDNA was replaced with the 4.4kb CFTR cDNA, integration efficiency dropped significantly. However, 1-2% integration of codon optimized donors was sufficient to restore CFTR expression in the bulk edited population of human bronchial epithelial cell line, 16HBE14o- (16HBE), to levels reaching 50% of wildtype expression as measured by Western Blot. Electrophysiological validation of CFTR ion channel function measured via Ussing Chamber Assay revealed that these bulk edited populations exhibit greater than 40% restoration of the chloride ion currents of the measured wildtype controls. These results demonstrate that low levels of CFTR integration can be made therapeutically relevant by optimizing the designs of gene editing reagents. Importantly, this work utilizes nonviral editing reagents, an essential step towards in vivo gene therapy for CF.

In the ciliate Tetrahymena , telomeres of the germline micronucleus (MIC) are removed and replaced by de novo telomere addition during somatic macronuclear (MAC) development. In this study, we investigated the kinetics and mechanism of the MIC telomere elimination. Comparison of the MIC and MAC genome sequences indicated that the MIC telomeres are excised from chromosomes as part of larger MIC-limited sequences (MLSs) through chromosomal breakage. We confirmed this using an optimized oligo-FISH protocol and found that their elimination occurs in parallel with other programmed DNA elimination processes. CRISPR-Cas9 disruption of a MLS-associated Chromosome Breakage Sequence (CBS) showed that elimination of the MLS was not blocked but instead led to loss of adjacent MAC-destined sequence (MDS), suggesting abnormal co-elimination. In biparental crosses of the CBS mutant, however, both MLS and MDS were retained, DNA elimination was broadly disrupted, and no viable progeny were produced. These findings indicate that chromosome breakage at MLS-associated CBSs is essential for the proper separation of MLSs and MDSs, ensuring correct DNA elimination and successful sexual progeny development. We propose that the MIC telomere elimination is subsumed within the broader process of programmed DNA elimination.

The epithelial sodium channel (ENaC) is essential for sodium reabsorption and potassium homeostasis in the distal nephron, where its activity is controlled by mineralocorticoid signaling and downstream proteolytic processing of channel subunits. While cleavage of the γ-ENaC subunit has been implicated in aldosterone-mediated sodium transport, the identity of mineralocorticoid receptor (MR)-regulated proteases responsible for this process remains uncertain. Here, we investigated the role of kallikrein-1 (encoded by Klk1 ), a serine protease expressed in the connecting tubule and cortical collecting duct (CNT/CCD), as a mediator of ENaC activation. Using CRISPR/Cas9, we generated a conditional Klk1 -floxed allele and established mice with CNT/CCD-specific deletion of Klk1 by crossing with Calb1 -Cre (CNT- Klk1 -/- ). On a low sodium, high potassium diet, CNT- Klk1 -/- mice exhibited ∼85% less renal kallikrein-1 expression, yet maintained normal serum electrolytes, urinary potassium excretion, and aldosterone responses. Western blot analysis revealed significantly less cleavage of γ-ENaC and α-ENaC in CNT- Klk1 -/- kidneys, accompanied by more total NCC abundance. Despite impaired ENaC proteolysis, amiloride-sensitive sodium excretion was preserved, indicating intact ENaC function. These findings identify renal kallikrein-1 as a protease that contributes to ENaC subunit processing in vivo . However, the absence of overt sodium or potassium handling defects in CNT- Klk1 -/- mice suggests that kallikrein-1 deficiency is not sufficient to disrupt overall ENaC function, likely due to compensatory mechanisms from redundant proteolytic or non-proteolytic pathways. Together, our results refine the role of kallikrein-1 as a modulator, rather than a sole determinant, of ENaC activation and highlight the complexity of aldosterone-dependent sodium transport in the distal nephron. New & Noteworthy Using a novel connecting tubule / cortical collecting duct specific kallikrein-1 knockout model, we show that γ- and α-ENaC cleavage is impaired by loss of renal kallikrein-1 without major disturbances in sodium or potassium handling. These findings highlight redundancy among ENaC regulatory pathways and suggest that proteolytic cleavage, while biochemically evident, may not be an accurate marker of ENaC-mediated sodium transport under physiological stress.

ABSTRACT Cancer cell evasion of therapy is a highly adaptive process that undermines the efficacy of many treatment strategies. A significant milestone in the study of these mechanisms has been the advent of pooled CRISPR knockout screens, which enable high-throughput, genome-wide interrogations of tumor dependencies and synthetic lethal interactions, advancing our understanding of how cancer cells adapt to and evade therapies. However, the utility of this approach diminishes when applied to dynamic biological contexts, where processes are transient and sensitivity to routine cell culture manipulations that introduce noise and limit meaningful discoveries. To overcome these limitations, we present RESTRICT-seq, a next-generation pooled screening methodology that restricts Cas9 nuclear activation in controlled, repeated cycles. By confining Cas9 catalytic activity to strict temporal windows, RESTRICT-seq mitigates undesired fitness penalties that routinely accumulate throughout pooled screens. When benchmarked against conventional pooled screens and standard inducible protocols, RESTRICT-seq revealed significantly fewer divergent cell clones and increased signal-to-noise ratio, overcoming a key limitation of traditional methods. Leveraging RESTRICT-seq, we conducted a comprehensive functional survey of the druggable mammalian epigenome, uncovering several elusive epigenetic drivers of treatment resistance in cutaneous squamous cell carcinoma (cSCC). This revealed PAK1 as a previously unrecognized mediator of cSCC resistance in human and mouse SCC, offering new insights into a prognostic marker and therapeutic target of high clinical significance. Our findings establish RESTRICT-seq as a powerful tool for extending the applicability of pooled CRISPR screens to dynamic and previously intractable biological contexts.