Abstract Genome-wide sequence analysis has identified the SPI1 gene as a genetic risk factor for Alzheimer's disease (AD). SPI1 encodes the PU.1 protein, which plays a critical role in microglial development and immune responses, primarily studied in mouse models. However, no studies have yet reported the impact of the SPI1 gene on AD-related phenotypes in zebrafish. Therefore, this study utilized CRISPR/Cas9 gene editing to generate spi1a knockout zebrafish mutants, investigating the effects of spi1a loss-of-function on AD-associated phenotypes. The results showed that spi1a knockout led to reduced locomotor activity and increased brain cell apoptosis in larvae, while working memory, acetylcholinesterase (AChE) activity and Aβ1–42 levels remained unchanged. In contrast, adult spi1a knockout zebrafish exhibited significant cognitive decline, upregulated apoptosis-related genes, elevated AChE activity and increased Aβ1–42 accumulation. Transcriptomic analysis further revealed that spi1a knockout altered the expression of multiple AD-related genes and affected immune and inflammation-related signaling pathways. In conclusion, spi1a deficiency induced AD-like phenotypes in adult zebrafish. This study demonstrates the role of spi1a in modulating AD-related phenotypes in both larval and adult zebrafish, providing crucial insights into AD pathogenesis and establishing a valuable model for future high-throughput drug screening and therapeutic development.

Focal adhesions (FAs) are dynamic macromolecular assemblies that anchor the actin cytoskeleton to the extracellular matrix via integrin receptors, thereby regulating cell morphology and migration. Although FA maturation and organization have been extensively studied, it remains unclear how regulatory proteins influence the 3D architecture of FAs. Here, we show that loss of the vasodilator-stimulated phosphoprotein (VASP) impairs adhesion dynamics. We employed CRISPR/Cas9-mediated knockout of VASP and/or the mechanosensitive adaptor protein zyxin to investigate their respective roles in actin–adhesion coupling. Loss of VASP and zyxin correlates with altered FA morphology and impaired dynamics. Using cryo-electron tomography (cryo-ET), we resolved the polarity of individual actin filaments associated with FAs and identified a contractility-related actin layer enriched with tropomyosin. VASP and zyxin are required for the assembly of dense and aligned actin bundles with uniform polarity, oriented with their barbed ends towards the cell edge. In contrast, the tropomyosin-decorated dorsal actin layer remains unaffected by these perturbations. Our findings reveal distinct, layered architectures within FAs and underscore the cooperative role of VASP and zyxin in stabilizing the organization of actin filaments at functional adhesion sites.

Predatory bacteria are abundant in soil, but their diversity and functions remain not fully understood, especially in subarctic regions. Here, we report strain 1-FT3.2, a predatory bacterium obtained from peatland soil in Northern Finland (Pallas, 68 °N). The bacterium was cultivated on Mucilaginibacter cryoferens FT3.2 as prey. Although a pure culture of strain 1-FT3.2 was not obtained, its draft genome was assembled from sequencing reads derived from the co-culture with its prey. The draft genome of 1-FT3.2 is 7.2 Mb in length and 81% complete. Genome analyses suggested that 1-FT3.2 belongs to the family Polyangiaceae (phylum Myxococcota ), which comprises predatory bacteria. The genome annotation revealed (near-)complete metabolic modules of central carbon metabolism and aerobic respiration. Two proviral regions were predicted in the draft genome, both putatively representing tailed phages of the class Caudoviricetes . Several CRISPR-Cas system proteins were also identified. The draft genome sequence could be used in future comparative studies assessing the diversity of predatory bacteria in northern soils or other environments.

The development of CRISPR-Cas9 cleavage activity prediction tools hinges on data produced from high-throughput guide-target lentiviral library screens for different Cas9 variants. However, the majority of such tools remain limited to predictions for one or few Cas9 variants, making it difficult to quantify the effects of Cas9 residues on cleavage activity. To bridge the gap, we introduce 4 interpretable DeepEmbCas9 models for the cleavage activity prediction of 40 type II-A and II-C Cas9 variants — DeepEmbCas9, DeepEmbCas9-MVE, DeepEnsEmbCas9 naive, and DeepEnsEmbCas9 — leveraging protein and RNA language model embeddings to encode Cas9 and sgRNA, respectively. Among the 4 neural network models, DeepEnsEmbCas9 naive performed the best in both in-distribution and out-of-distribution settings, where DeepEnsEmbCas9 naive outperformed individual Cas9 cleavage activity prediction tools on 18 out of 51 and 17 out of 48 benchmark test sets, respectively, and performed comparably otherwise. Concerning uncertainty quantification, DeepEnsEmbCas9 yields quantile-calibrated uncertainty estimates while keeping a minimal performance drop compared to DeepEnsEmbCas9 naive. SHAP importance analysis on DeepEmbCas9 reaffirms the importance of Cas9-target PAM binding as a first step for Cas9 cleavage, and reveals the L2 linker and PLL-WED-PI as important Cas9 domains modulating DeepEmbCas9’s predicted activity change when introducing increased-fidelity and PAM-altering Cas9 mutations, respectively. Our findings demonstrate the usefulness of protein language model embeddings in uncertainty-aware Cas9 cleavage activity prediction. More generally, DeepEmbCas9 models serves as an initial step towards cleavage activity prediction modelling for the whole Cas9 protein family.

Gene drive can control pathogen transmission or suppress vector populations by spreading drive alleles with super-Mendelian inheritance. CRISPR homing drive currently represents the most powerful type, and regulating Cas9 expression with specific promoters has been effective for improving drive performance. However, selecting these is often a major challenge. Here, we evaluated 35 Cas9 constructs driven by distinct promoters in different gene drive systems and identified associations between drive performance and single-cell RNA expression patterns of the promoter-associated genes. Our results indicate that higher drive conversion is significantly associated with elevated expression of the promoter-associated gene in the respective reproductive cells, but embryo resistance allele formation correlates with excessive female germline expression. For males, early germline expression produces superior performance. Thus, we find that optimal drive performance requires restricting Cas9 expression to a tight quantitative and spatiotemporal window. In addition, found that in situ integrated rhino -Cas9 constructs significantly reduce somatic expression, underscoring the importance of genomic locus. On the basis of these results, we propose criteria for selecting promoters, providing a theoretical rationale and practical guidance for optimization of promoter elements in homing gene drive systems.

Current models of microRNA (miRNA) silencing posit that RNA-sequence rules are sufficient for canonical targeting of mRNAs by Argonaute 2 (AGO2), the central protein of the miRNA-induced silencing complex (miRISC). Using chimeric eCLIP in CRISPR-edited LIMD1 +/+ , LIMD1 +/− , and LIMD1 −/− human small airway epithelial cells (hSAECs), we reveal a transcriptome-wide dependency on LIMD1, an AGO2 adaptor, for effective miRNA targeting and repression. In LIMD1-deficient cells, miRNA loading is uncoupled from productive targeting: despite increased AGO2–miRNA interactions, complexes engage fewer transcripts and sites, reducing occupancy and more than halving both the breadth and depth of targeting. We also observe altered AGO2 positional footprints across targets in LIMD1-deficient cells. LIMD1 dependence is most pronounced at defined RNA contexts: weak (GC-poor) seed pairings, interactions involving evolutionarily young miRNAs or sites that nonetheless form thermodynamically stable duplexes, with these losses particularly enriched in coding sequences of rapidly evolving C 2 H 2 -zinc-finger genes. Even within canonical seed repertoires of individual AGO2–miRNAs, LIMD1 is most critical at poorly conserved sites, indicating that LIMD1 broadens miRNA regulation beyond ancient, deeply conserved targets. In culture, LIMD1 deficiency de-represses oncogenic proteins that, in vivo , inversely correlate with LIMD1 levels in normal lung and adenocarcinoma, where LIMD1 is characteristically reduced, and whose dysregulation predicts poor survival. Thus, LIMD1 emerges as a key determinant of miRISC architecture, targeting, and potency, challenging RNA-centric models of miRNA function and exemplifying how adaptor proteins diversify post-transcriptional regulation. Graphical Abstract LIMD1 defines the scope of miRNA-mediated targeting and repression AGO2-chimeric eCLIP in CRISPR-edited human small airway epithelial cells (hSAECs) shows LIMD1 is required for productive AGO2–miRNA engagement transcriptome-wide. LIMD1 deficiency reduces the AGO2–miRNA:targetome. Each AGO2–miRNA binds fewer targets, with lower occupancy per site and per transcript and fewer global silencing events. LIMD1 dependence is strongest for GC-poor seed-sites, less conserved miRNAs and sites, and thermodynamically stronger duplexes. Dosage-dependent effects of LIMD1 deficiency are broadly observed. Target-mRNA decay and translational repression are reduced in LIMD1-deficient hSAECs, increasing protein output. In vivo , LIMD1 LOH–associated deficiency is prevalent and typically clonal in NSCLC. LIMD1 expression inversely correlates with oncogene levels in normal lung and adenocarcinoma, and target dysregulation predicts poor survival.

Summary Single-stranded DNA (ssDNA) gaps are a hallmark of BRCA-deficient cells, yet the mechanisms that safeguard these lesions remain unclear. Through a genome-wide CRISPR screen, we identified the RAD9A-HUS1-RAD1 (9-1-1) complex as essential for the survival of BRCA2-deficient cells through an ATR-independent mechanism. Loss of 9-1-1 in this context leads to the accumulation of PRIMPOL-dependent gaps that fail to undergo post-replicat ive repair, resulting in pathological expansion and increased DNA damage. This instability is driven by excessive EXO1-mediated degradation, as EXO1 depletion rescues the phenotype. We further demonstrate that the 9-1-1 complex is required for POLζ-dependent gap filling. We propose a model in which ssDNA gaps, when extended beyond a critical length, become inaccessible to TLS-mediated repair and are fully reliant on homologous recombination. These findings establish the 9-1-1 complex as key regulator of ssDNA gap stability and a promising therapeutic target in BRCA2-deficient cancers. Graphical Abstract

Chlamydia is an obligate intracellular bacterium that differentiates between infectious, non-dividing EBs and non-infectious, dividing RBs. Pathogenic Chlamydia species are unusual in lacking a peptidoglycan sacculus, yet they do synthesize a transient and localized peptidoglycan structure at the divisome of the RB during their polarized division process. Although several studies have described the components of the chlamydial divisome necessary to generate peptidoglycan at a specific site on the membrane, less is understood about how the peptidoglycan structure is degraded to allow for the daughter cell to form and the division process to complete. Amidases are key components of the cell wall in model system bacteria as they catalyze the degradation and remodeling of peptidoglycan, including in the division septum. Here, we characterized the cell division-associated amidase, AmiA_Ct, of Chlamydia trachomatis both in vitro and in vivo . Our in vitro data show that AmiA_Ct is a bona fide , metal-dependent amidase capable of cleaving peptidoglycan. AmiA_Ct complemented an E. coli amidase deficient strain and supported the growth and separation of daughter cells. To assess the function of AmiA_Ct in C. trachomatis , we generated a transformant strain carrying an inducible CRISPR interference system targeting the amiA gene. Knocking down expression of amiA resulted in altered bacterial morphology, a reduction in infectious EBs, and the accumulation of peptidoglycan in the organisms. These data indicate a critical function for AmiA_Ct in the unique cell division process of Chlamydia . Importance Peptidoglycan is an important structural cell wall polymer that serves to give bacteria their shape and resistance to changes in extracellular solute concentrations. For Chlamydia trachomatis , an obligate intracellular pathogen that divides within a host cell, peptidoglycan is only used for cell division and is not a component of its cell wall. In this study, we characterize the function of a chlamydial amidase that helps degrade peptidoglycan during cell division. We show a critical function for amidase activity in facilitating changes to the peptidoglycan structure during chlamydial cell division that support normal growth and development of this pathogenic bacterium.

Toxoplasma gondii, the causative agent of toxoplasmosis widespread in animals and humans, is an intracellular apicomplexan protozoan parasite infecting a variety of host cells. Gene editing using CRISPR-Cas9 has become a standard tool to investigate the molecular genetics of this interaction. With respect to gene knock-out (KO) studies, the general paradigm implies that the gene of interest is expressed in the wildtype and that only the gene of interest is affected by the knock-out. Consequently, the observed phenotype depends on presence or absence of genes of interest. To challenge this paradigm, we knocked out two open reading frames (ORFs) constitutively expressed in T. gondii ShSp1 tachyzoites, but not essential, namely ORF 297720 encoding a trehalose-6-phosphatase homolog and ORF 319730 encoding a You2 C2C2 zinc finger homolog. We analyzed the proteomes of tachyzoites isolated at a late stage of infection, of intracellular tachyzoites and of host cells at an early stage of infection. The intended KO proteins were present in the T. gondii Sp1 wildtype but absent in the KO clones. Moreover, besides differentially expressed (DE) proteins specific to each KO, 17 DE proteins common to both KOs were identified in isolated and 39 in intracellular tachyzoites. Moreover, 76 common DE proteins were identified in host cells. Network and enrichment analyses showed that these proteins were functionally related to antiviral defense mechanisms. These results indicate that the KO of a gene of interest may not only affect the expression of other genes of the target organism, in our case T. gondii, but also the gene expression of its host cells. Therefore, phenotypes of KO strains may not be causally related to the KO of a given gene.

Natural killer (NK) cells are emerging as a promising platform for engineered adoptive cell therapies. However, gene editing in NK cells remains challenging, and more effective strategies are needed. Here, we established a robust, feeder-free, and modular workflow for genome engineering in primary human NK cells, combining CRISPR/Cas9 with AAV6-mediated transgene delivery. Efficient site-specific transgene integration was achieved at various loci and can be coupled with concurrent disruption of the target locus in a single editing step. Furthermore, transgene expression was tunable according to the integration site and promoter. We applied this strategy to target a chimeric antigen receptor (CAR) transgene to a panel of inhibitory NK receptor loci, establishing a synergistic approach to enhance anti-tumor activity and facilitate the reliable comparison of CAR variants without expression bias. We identified TIGIT as an ideal locus that supports strong CAR expression and anti-tumor function. This genome engineering framework, which leverages multiple, complementary and precisely controlled genetic edits, can support the rational design of future NK-cell therapies tailored to overcome cell-intrinsic limitations and tumor-specific barriers.

The budding yeast Saccharomyces cerevisiae is a central model organism in genetics and synthetic biology, yet efficient multiplex genome editing remains difficult because many toolkits are restricted by limited plasmid selection markers and reduced efficiency when targeting multiple loci. In our previous work, we introduced a CRISPR-based system incorporating three nucleases with distinct PAM specificities but it was available only with the URA3 marker. Here we present LOBSTERS, an expanded modular vector series that retains the PAM-diverse nucleases (SpCas9, SaCas9, and enAsCas12a) while extending marker options to seven. This design enables the simultaneous use of multiple plasmids in one transformation, supporting scalable and flexible genome editing. Proof-of-concept experiments demonstrated efficient ADE2 and ADE3 deletions with colorimetric readouts, coordinated tagging of essential proteins (Cdc3 and Cse4) without compromising function, and recapitulation of three quantitative trait variants ( RME1, TAO3 , and MKT1 ) underlying sporulation efficiency. Together these results establish LOBSTERS as a robust and versatile platform for multiplex genome editing in S. cerevisiae . By enabling coordinated modification of essential proteins, genetic interactions, and quantitative trait variants, LOBSTERS provides a broadly applicable resource for functional cell biology and synthetic biology in yeast. Significance Statement LOBSTERS integrates three PAM-diverse nucleases with seven plasmid selection markers, overcoming the single-marker limitation of previous yeast genome editing toolkits. The system enables efficient simultaneous editing at multiple loci, demonstrated by functional tagging of essential proteins and recapitulation of quantitative trait variants. By broadening the editable space and lowering barriers to complex genotype construction, LOBSTERS provides a widely applicable resource for yeast cell biology and synthetic biology.

Generation of arrayed genome-wide CRISPR libraries in a ready-to-transduce lentiviral format remains laborious, time-consuming, and costly. To address these limitations, the present study developed a fully automated lentivirus production and titration workflow using a Biomek i7 Hybrid automated workstation, integrated with multiple instruments and managed by SAMI EX software. The workflow produced and titrated viruses in 96 and 384-well plate formats, respectively. It employed reverse transfection and triplicate wells per lentivector to reduce variability and yielded an average of three viral particles in transduction unit (TU) per producing HEK293T cell. Titration was performed using U937-mCherry suspension cells, with the percentage of transduced cells converted from U937 (X%) to HEK293T (Y%) values via a linear regression equation (Y% = 4.3X% + 9.3%). The titer calculation was based on the initial seeding cell number, the converted percentage of HEK293T transduced cells, and virus input volume. The titration demonstrated strong reproducibility across LSRFortessa (BD) and Aurora (Cytek) flow cytometers (R 2 = 0.9). Among 1,760 unconcentrated virus preparations, median and mean titers reached approximately 1.2 x 10 6 TU/mL, with over 97% of samples exceeding the high-titer threshold of 2x10 5 TU/mL, thus demonstrating a robust, scalable, and cost-effective automation platform for high throughput arrayed lentiviral library production and titration.

The myeloid oncogene TRIB2 is a key driver of acute myeloid leukaemia (AML) pathogenesis, promoting chemoresistance and blocking differentiation through ubiquitin-mediated degradation of the C/EBPα transcription factor. Despite its stable and sometimes elevated expression across AML subtypes, TRIB2 remains a clinically-untargeted vulnerability. Here, we present a comprehensive investigation into TRIB2 degradation mechanisms using multimodal approaches, including CRISPR knockout, mutational protein stability, small molecule TRIB2 engagement and evaluation of a novel targeted protein degrader (TRIB2-PROTAC). We identify Afatinib, a multi-ERBB covalent inhibitor, as a rapid inducer of TRIB2 degradation, triggering AML cell death via an ERBB-independent pathway. Importantly, TRIB2 degradation synergized with cytarabine, the frontline AML chemotherapy, amplifying therapeutic efficacy. Mapping of TRIB2 ubiquitination sites revealed Lys-63 as critical for its own proteolytic turnover, and a Lys to Arg degradation-resistant mutant (K all R) conferred enhanced chemoresistance and increased leukaemic engraftment in vivo . CRISPR-mediated TRIB2 knockout validated an essential role in AML cell survival. Consistently, the novel TRIB2-PROTAC (compound 5K) achieved robust TRIB2 degradation and AML cell killing at low micromolar concentrations. These findings establish TRIB2 as a compelling therapeutic target in AML and demonstrate that leveraging the ubiquitin-proteasome system to degrade TRIB2 offers a promising strategy to overcome chemoresistance. This work provides strong preclinical rationale for the development of TRIB2-targeting therapies in AML.

Plant immunity mediated by nucleotide-binding leucine-rich repeat (NLR) receptors often relies on canonical EDS1 / NDR1 signaling, but alternative mechanisms are emerging. We uncover a novel modular, noncanonical immune hub orchestrated by Rcr1 , a TIR-NLR (TNL) gene conferring clubroot resistance in Brassica napus against the root-infecting protist Plasmodiophora brassicae . Unlike typical TNLs, Rcr1 engages non-NLR partners in two separable modules: a CP1 (cysteine protease)–WRKY-based recognition module, likely monitoring a pathogen virulence target, and an AP (ankyrin-repeat protein)–ERF-based signaling module, driving jasmonic acid/ethylene-mediated defense. This architecture functions without detectable EDS1 / NDR1 involvement, challenging salicylic acid-dominant models of biotrophic immunity and expanding current views of how TNLs can be wired in plant defense. Using high-throughput interactor screening and CRISPR/Cas9 knockouts, we validate these modules, while heat-inducible gene excision reveals Rcr1 ’s critical early role (0–14 days post-inoculation). Together, our findings position Rcr1 as an exemplar of modular TNL architecture, suggesting that separable recognition and signaling branches may represent a broader principle of immune flexibility in plants. This study redefines TNL flexibility, offering a blueprint for breeding durable disease-resistant crops via modular immune engineering, with clubroot resistance as a model.

Only a fraction of bacterial genomes encode CRISPR-Cas systems but the selective causes of this variation are unexplained. How naturally virulent bacteriophages (phages) select for CRISPR immunity has rarely been tested experimentally. Here, we show against a panel of genetically and functionally diverse virulent phages that CRISPR immunity was not universally beneficial, and its fitness effect varied strongly between phages in predictable ways. In addition to mechanisms known to alter the effectiveness of CRISPR immunity, such as encoding a matching spacer or a protective nuclear shell, we show that the fitness effect of CRISPR immunity negatively correlated with the probability of evolving receptor-based resistance to the phage via spontaneous mutation. Supply of resistance mutations differed strongly between very closely related lipopolysaccharide-binding phages and was associated with variation at the C-terminus of the tail fibre protein altering residues involved in hydrogen bonding and the predicted binding site. Our results show that CRISPR immunity is more beneficial against virulent phages that are harder to evolve resistance to via receptor mutations, suggesting that virulent phage community composition and diversity will be important drivers of the prevalence of CRISPR immunity.

Influenza A virus (IAV) causes major economic losses to the poultry industry and poses a zoonotic threat to human health. Potential pandemic outbreaks are underpinned by the ability of the virus to jump from one species to another. Host-virus interactions can dictate the success of such events and while systematic studies have successfully mapped host virus interactions in human cells, few studies have been performed in relevant animal host cell lines. Here, we conducted two independent genome-wide CRISPR/Cas9 knockout screens in chicken lung epithelial cells infected with either the human-adapted PR8 vaccine strain or the avian UDL 3:5 reassortant virus encoding PR8 HA, NA and M segments. Rather than selecting solely for cell survival, we used anti-M2 antibody staining and fluorescence-activated cell sorting to capture host factors influencing multiple stages of the IAV life cycle. Across both screens, we identified 104 genes required for efficient replication in chicken cells, including 16 with strong effects (log₂ fold change > 2). Comparative analysis with published human screens revealed 17 conserved host factors, 19 human-specific factors, and 42 chicken-specific factors, highlighting potential species-specific interactions. Top hits included genes involved in sialic acid biosynthesis and N-linked glycosylation— SLC35A1 , SLC35A2 , and the avian-specific influenza polymerase cofactor ANP32A . Functional validation demonstrated that MOGS , MGAT1 , DENR , DMXL1 , ENO1 , IPO9 , KLF6 , PTAR1 , and TSG101 contribute to multiple stages of the IAV life cycle. In particular, MOGS and MGAT1 were essential for N-glycan processing and modulated cell-surface sialic acid abundance, with strain- and species-specific effects. These findings define a genetic landscape of IAV dependency factors in chicken cells and suggest shared and species-specific host requirements that could impact cross-species transmission.

Genetic perturbations are one of the great strengths of the model organism Drosophila melanogaster , with approaches such as classical mutagenesis and RNA interference enabling a wealth of biological discoveries. A more recent approach for altering gene expression is CRISPR/Cas9-based mutagenesis, but as with any new tool, its use must be optimized. High expression of Cas9 has been shown to cause cytotoxicity in some cell types. Here, we show that Cas9 expression alone causes cytotoxicity in the dendritic arborization (da) neurons that are widely used to study neuronal development and regeneration. We then systematically evaluate alternative Cas9 transgenes designed to lower total Cas9 expression, called uCas9 transgenes. We show that expression of these uCas9 transgenes results in little to no cytotoxicity to da neurons. Lastly, we demonstrate the ability of uCas9 transgenes to effectively and specifically gene edit in da neuro ns. Thus, we expand the toolkit of genetic perturbations available to researchers working with Drosophila da neurons or other cell types suceptible to cytotoxicity due to high expression of Cas9.

ABSTRACT Nicotine is a plant-derived pyridine alkaloid with potent neurotoxic properties. A major pathway for detoxification of nicotine in mammals is via glucuronidation to produce nicotine N -glucuronide, but this process in insects remains poorly understood. Using mass spectrometry, we demonstrate that Drosophila melanogaster detoxifies nicotine through glycosylation, producing nicotine N -glycoside. Given that many new agrochemicals contain pyridine rings, we also investigated the metabolism of flonicamid and imidacloprid. We detected glycosylation of flonicamid, but not imidacloprid. A targeted RNAi screen across 21 UDP-glycosyltransferases ( Ugt s) identified Ugt35B1 as important for survival of nicotine exposure. CRISPR-based knockout of Ugt35B1 increases sensitivity to nicotine and flonicamid, but not to imidacloprid, nor to a structurally distinct neonicotinoid (thiamethoxam). Mass spectrometry of knockout and control flies confirms that Ugt35B1 glycosylates nicotine, its metabolite cotinine, and flonicamid. Together these findings establish Ugt35B1 as the principal UGT mediating nicotine detoxification in D. melanogaster , revealing a previously uncharacterized insect glycosylation pathway with potential implications for herbivory, insecticide detoxification and toxicology. Highlights - Drosophila detoxifies nicotine by glycosylation into nicotine N -glycoside. - A targeted RNAi screen identifies Ugt35B1 as critical for nicotine survival. - Ugt35B1 knockout sensitizes flies to nicotine and flonicamid, but not to imidacloprid or thiamethoxam. - First demonstration of an insect UGT mediating in vivo glycosylation of nicotine and cotinine.

The Fascin family of actin-bundling proteins organizes actin filaments (F-actin) into tightly packed bundles that drive dynamic membrane protrusions such as filopodia. In neurons, fascin has been thought to primarily function in axons, as previous studies reported its absence from dendritic filopodia and spines. Here, we demonstrate that fascin is both present and functionally important in dendritic compartments. Using optimized immunocytochemistry and CRISPR-based endogenous tagging of fascin1 in cultured hippocampal neurons, we show that fascin localizes to developing dendritic filopodia and is enriched in mature dendritic spines. Super-resolution imaging further reveals that fascin is organized into discrete nanoscale foci within spine heads, but not the spine neck. Finally, we show that CRISPR-mediated knockout of fascin1 in mature hippocampal neurons impairs synaptic potentiation, without affecting baseline excitatory synaptic transmission. Together, our findings uncover a previously overlooked aspect of actin organization in dendritic spines and establish fascin as a critical regulator of postsynaptic plasticity. Summary Statement The actin bundling protein fascin localizes to dendritic filopodia and spines, where it regulates activity-dependent synaptic plasticity.

ABSTRACT Chromosome segregation during anaphase occurs through two mechanistically distinct processes: anaphase A, in which chromosomes move toward spindle poles, and anaphase B, in which the anaphase spindle elongates through cortical astral microtubule pulling forces. Caenorhabditis elegans embryos have been thought to rely primarily on anaphase B, with little to no contribution from anaphase A. Here, we uncover a novel anaphase A mechanism in C. elegans embryos, driven by the kinesin-13 KLP-7 MCAK and opposed by the kinesin-12 KLP-18. We found that the extent of chromosome segregation during anaphase A is asymmetrically regulated by cell polarity cues and modulated by mechanical tension within the spindle, generated by opposing forces acting on chromosomes and spindle poles. Additionally, we found that the contribution of anaphase A to chromosome segregation increases progressively across early embryonic divisions. These findings uncover an unexpected role for anaphase A in early C. elegans development and reveal a KLP-7 MCAK -dependent mechanical coordination between anaphase A and anaphase B driven chromosome segregation. eTOC summary Dias Maia Henriques et al. uncover an anaphase A pathway, driven by the kinesin-13 KLP-7 and opposed by the kinesin-12 KLP-18, that contributes to chromosome segregation in early C. elegans embryos. Its activity is regulated by spindle tension, cell polarity cues, and progressively increases during early embryonic divisions.

In vertebrates, vitamin A (VA) is crucial for development, tissue homeostasis, vision, and immunity. Retinal, a form of VA, is produced via enzymatic cleavage of β-carotene by beta-carotene oxygenase 1 ( bco1 ) and bco1-like ( bco1l ). While bco1 is found across vertebrate taxa, bco1l is a paralog of bco1 that we discover to have evolved in the ray-finned fishes, the most abundant, speciose, and commercially important group of fishes. We investigated the function of bco1l in ray-finned Siamese fighting fish, commonly known as betta, an emerging model for genetics and development. Using CRISPR-Cas9 knockouts, we find that lack of bco1l results in reduced VA and elevated β-carotene in larvae, starting when animals have exhausted their yolk supply of retinal, followed by stunted growth and death during juvenile development. Exogenous retinoic acid rescues the mutation, demonstrating its deficiency causes these defects. bco1l is 4× more abundant than bco1 in the intestine. This, coupled with the inability of bco1 to sustain VA production in the bco1l mutant, indicates that bco1l is the primary enzyme for dietary carotenoid conversion into retinal. Our results show that VA production by bco1l is required for post-embryonic development, and that bco1l became essential after evolving via duplication of bco1 .

Homologous recombination (HR) is ubiquitous across evolution, driving adaptation by reshuffling standing genetic variation. Although bacteria lack meiotic recombination, HR extensively shapes their genomes. However, the mechanisms and ecological conditions sustaining frequent HR in bacteria remain unclear. Using Escherichia coli , we reveal how frequent recombination emerges from herd immunity to a generalized transducing phage. Herd immunity–established here via CRISPR immunity–maintains genetic polymorphism and enables stable host–phage coexistence, thereby promoting genome-wide gene flow and accelerating adaptation through recombination up to two orders of magnitude relative to de novo mutations. Notably, we show that recombination occurs in stationary phase and is mediated by RecG, which has been previously reported to be regulated by the stringent response – a bacterial reaction to nutrient deprivation and other stress conditions. Bacterial herd immunity thus fulfills an unexpected role of promoting adaptation by HR. This mechanism helps explain the enigmatic high rates of HR across bacterial populations, clarifies how bacteria adapt as resources wane, and suggests a broader evolutionary role for bacterial immune systems beyond individual defense.

ABSTRACT Plasmids are a foundational research reagent and a key material in biopharmaceutical manufacturing. Plasmids require a backbone for propagation in E. coli, which typically contains an antibiotic resistance gene alongside a replication origin. As plasmids increasingly enter clinical applications, concerns are raised on the safety risks of antibiotic resistance genes. Additionally, these protein-coding genes occupy long stretches of DNA that incur significant metabolic burdens on host cells, which negatively impacts plasmid manufacturability and functionality, leading to high production cost and compromised clinical efficacy. Here, we describe miniVec, a novel miniaturized plasmid backbone devoid of protein-coding sequence, and instead expresses a small RNA to provide constant selective pressure capable of sustaining high plasmid copy numbers in plain culture media devoid of antibiotics or other chemical additives. This simplifies large-scale fermentation and greatly increases plasmid yield. Notably, miniVec confers enhanced functionality in a variety of applications such as chemical transfection, electroporation, virus packaging, transposon-or CRISPR-mediated genome integration, and in vivo naked DNA transfection and vaccination, while exhibiting no detectable immunogenicity or toxicity. These advantages establish miniVec as the next-generation plasmid platform for clinical applications, featuring improved safety that aligns with regulatory expectations, enhanced manufacturability leading to much higher yield and dramatic cost reduction, and augmented functionality in diverse applications.

ABSTRACT Dravet syndrome (DS) is a severe childhood epilepsy caused by mutations of the sodium channel NaV1.1. These mutations are thought to compromise the ability of inhibitory interneurons to regulate network activity, leading to seizure events. However, standard treatments to restore inhibition have limited efficacy, suggesting the existence of additional pathological mechanisms. Here, we use hiPSC-derived neuronal networks containing both excitatory and inhibitory neurons to show that excessive bursting activity in DS cultures is driven by excitatory neurons. This rise in bursting frequency is caused by the increased expression of HCN1 “pacemaker” channels in excitatory neurons, and bursting activity can be normalised using a channel blocker. With this work, we propose a new pathophysiological mechanism in DS and identify HCN1 as a novel therapeutic target.

ABSTRACT Neuronal ceroid lipofuscinosis type 2 (CLN2) is a rare, fatal paediatric neurodegenerative genetic disease that causes progressive psychomotor decline, epilepsy, speech impairment, vision loss, and premature death by late childhood or early adolescence. It is caused by mutations in the TPP1 gene, which encodes the tripeptidyl peptidase 1 enzyme. To date, enzyme replacement therapy (ERT) with recombinant human TPP1 is the only approved treatment available and it does not appear to have a curative effect on disease progression or rescuing complex motor functions. This underscores the importance of developing more effective treatment strategies for CLN2 disease. In the quest for an alternative therapy, we here seek to utilise CRISPR based prime editing (PE) to correct one of the most frequent TPP1 mutations of c.622 C>T (p. Arg208Ter). In cultured mammalian HEK293a cells we screened 16 different PE and pegRNA combinations, achieving on-target editing efficiencies up to 27.36 %. To enable efficient in vivo gene editing, we packaged the most promising PE and pegRNA combination using engineered virus-like particles (eVLPs) which resulted in 12.6 % on-target editing. Given the robust on-target editing and reduced risk of bystander editing, PE is deemed suitable for in vivo testing in the pre-clinical models for CLN2 disease. Overall, our findings establish the proof of concept for CRISPR-based prime editing to correct pathogenic human TPP1 nonsense mutation of c.622 C>T (p. R208X) and provide a basis for further investigations of PE as a genetic treatment for CLN2 disease.