ABSTRACT Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated proteins (Cas) systems have revolutionized genome editing by providing high precision and versatility. However, most genome editing applications rely on a limited number of well-characterized Cas9 and Cas12 variants, constraining the potential for broader genome engineering applications. In this study, we extensively explored Cas9 and Cas12 proteins and developed CasGen, a novel transformer-based deep generative model with margin-based latent space regularization to enhance the quality of newly generative Cas9 and Cas12 proteins. Specifically, CasGen employs a strategies that combine classification to filter out non-Cas sequences, Bayesian optimization of the latent space to guide functionally relevant designs, and thorough structural validation using AlphaFold-based analyses to ensure robust protein generation. We collected a comprehensive dataset with 3,021 Cas9, 597 Cas12, and 597 Non-Cas protein sequences from reputable biological databases such as InterPro and PDB. To validate the generated proteins, we performed sequence alignment using the BLAST tool to ensure novelty and filter out highly similar sequences to existing Cas proteins. Structural prediction using AlphaFold2 and AlphaFold3 confirmed that the generated proteins exhibit high structural similarity to known Cas9 and Cas12 variants, with TM-scores between 0.70 and 0.85 and root-mean-square deviation (RMSD) values below 2.00 Å. Sequence identity analysis further demonstrated that the generated Cas9 orthologs exhibited 28% to 55% identity with known variants, while Cas12a variants show up to 48% identity. Our results demonstrate that the proposed Cas generative model has significant potential to expand the genome editing toolkit by designing diverse Cas proteins that retain functional integrity. The developed deep generative approach offers a promising avenue for synthetic biology and therapeutic applications, enableling the development of more precise and versatile Cas-based genome editing tools.

Clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)-based in vivo chromosomal rearrangements are a promising approach for generating model organisms with specific chromosomal abnormalities. However, conventional in vivo methods rely on viral vectors, which are expensive, require specialized equipment, and pose potential safety risks, thereby limiting their widespread application. To overcome the limitations above, we developed a novel, efficient, and cost-effective in vivo chromosomal engineering strategy using CRISPR ribonucleoprotein electroporation for the murine uterine epithelium. Our method successfully induced translocations at multiple loci and repaired a 57.8-Mb inversion. The findings of the present study establish in vivo electroporation as a practical alternative to traditional chromosomal engineering methods and provide a foundation for its broader application in genome editing technologies.

Abstract Klebsiella pneumoniae (Kp) has evolved as a major public health threat due to its multidrug-resistance (MDR) and hypervirulence. Current genome-editing tools for Kp are constrained by cumbersome workflows, low flexibility, and limited scalability. Here, we present the RECKLEEN system —Recombineering/CRISPR-based KLebsiella Engineering for Efficient Nucleotide editing — as a single plasmid platform designed for precise genetic manipulation of Kp. RECKLEEN combines lambda Red recombineering with powerful CRISPR-Cas9-based targeted counterselection, achieving up to 99.998% killing efficiency. By implementing the near PAM-less SpG Cas9 variant in RECKLEEN, the compatible target sequence spectrum was significantly broadened. This approach enables deletions, point mutations, and DNA integrations, with efficiencies reaching 100% of the counter-selected clones. Simultaneous multi-target deletions were accomplished with up to 72% efficiency. To streamline the process, we developed a toolbox of eleven plasmids based on a modular cloning standard, enabling time- and resource-efficient assembly of editing constructs. This allows a 5-days workflow, from plasmid construction to the generation of strains with the desired genetic modification(s). The efficacy of RECKLEEN extends to various MDR Kp strains, such as ATCC 700721, ATCC BAA-1705, and ATCC 700603, demonstrating its broad applicability. RECKLEEN significantly enhances genome-editing capabilities for Kp, advancing research into its pathology and MDR mechanisms.

Polygenic traits are expected to show high genetic redundancy and therefore low repeatability in the genomic response to selection. We tested this prediction by selecting for large body size in the black soldier fly ( Hermetia illucens ). Over three replicate experiments selected for large body size, we found a strong and repeatable phenotypic response, with a mean 15% increase in body size. Selected lines also increased in larval growth rate (+19%) and average protein content (+14%), suggesting that selection on large body size does not result in strong trade-offs. In contrast to the predictability of the phenotypic response across replicates, whole genome sequencing identified a highly polygenic and non-repeatable genomic response. We identified 120, 301 and 157 outlier genomic regions in the three replicates, but high redundancy with only four shared regions. Among 12 candidate genes found in these regions, the insulin-like receptor gene ( HiInR ) was confirmed as regulating larval growth using a CRISPR knockout experiment. In summary, polygenic quantitative traits show high genetic redundancy, even where the phenotypic response to selection is highly repeatable.

Escherichia coli is a ubiquitous gut commensal but also an opportunistic pathogen responsible for severe intestinal and extra-intestinal infections. Shiga toxin-producing E. coli (STEC) pose a significant public health threat, particularly in children, where infections can lead to bloody diarrhea and progress to hemolytic uremic syndrome (HUS), a life-threatening condition with long-term complications. Antibiotics are contraindicated in STEC infections due to their potential to induce prophages carrying Shiga toxin ( stx) genes, triggering toxin production. Here, we present a CRISPR-based antimicrobial strategy that selectively targets and eliminates O157 STEC clinical isolates while preventing toxin release. We designed a Cas12 nuclease to cleave >99% of all stx variants found in O157 strains, leading to bacterial killing and suppression of toxin production. To enable targeted delivery, we engineered a bacteriophage-derived capsid to specifically transfer a non-replicative DNA payload to E. coli O157, preventing its dissemination. In a mouse STEC colonization model, our therapeutic candidate, EB003, reduced bacterial burden by a factor of 3×10 3 . In an infant rabbit disease model, EB003 mitigated clinical symptoms, abrogated stx-mediated toxicity, and accelerated epithelial repair at therapeutically relevant doses. These findings demonstrate the potential of CRISPR-based antimicrobials for treating STEC infections and support further clinical development of EB003 as a precision therapeutic against antibiotic-refractory bacterial pathogens.

The rate, spectrum, and biases of mutations represent a fundamental force shaping biological evolution. Convention often attributes oxidative DNA damage as a major driver of spontaneous mutations. Yet, despite the contribution of oxygen to mutagenesis and the ecological, industrial, and biomedical importance of anaerobic organisms, relatively little is known about the mutation rates and spectra of anaerobic species. Here, we present the rates and spectra of spontaneous mutations assessed anaerobically over 1000 generations for three fermentative lactic acid bacteria species with varying levels of aerotolerance: Lactobacillus acidophilus, Lactobacillus crispatus, and Lactococcus lactis. Our findings reveal highly elevated mutation rates compared to the average rates observed in aerobically respiring bacteria with mutations strongly biased towards transitions, emphasizing the prevalence of spontaneous deamination in these anaerobic species and highlighting the inherent fragility of purines even under conditions that minimize oxidative stress. Beyond these overarching patterns, we identify several novel mutation dynamics: positional mutation bias around the origin of replication in Lb. acidophilus, a significant disparity between observed and equilibrium GC content in Lc. lactis, and repeated independent deletions of spacer sequences from within the CRISPR locus in Lb. crispatus providing mechanistic insights into the evolution of bacterial adaptive immunity. Overall, our study provides new insights into the mutational landscape of anaerobes, revealing how non-oxygenic factors shape mutation rates and influence genome evolution.

Investigating the temporal dynamics of gene expression is crucial for understanding gene regulation across various biological processes. Using the Fluorescent Timer protein (Timer), the Timer-of-cell-kinetics-and-activity (Tocky) system enables analysis of transcriptional dynamics at the single-cell level. However, the complexity of Timer data has limited its broader application. Here, we introduce an integrative approach combining molecular biology and machine learning to elucidate Foxp3 transcriptional dynamics through flow cytometric Timer analysis. We have developed a Convolutional Neural Networks (ConvNet) approach that incorporates image conversion and Gradient-weighted Class Activation Mapping (Grad-CAM) for class-specific feature identification at the single-cell level. Biologically, we developed a novel CRISPR mutant of Foxp3-Tocky lacking the Conserved Non-coding Sequence 2 (CNS2), which has successfully elucidated CNS2-dependent Foxp3 transcription dynamics, revealing novel roles of CNS2 in regulating Foxp3 transcription frequency under specific conditions. Furthermore, generating new data from WT Foxp3 Tocky mice at various ages, the Grad-CAM methods successfully revealed distinct dynamics of Foxp3 expression from neonatal to aged mice, highlighting prominent thymus-like features of neonatal splenic Foxp3 + T cells. In conclusion, our study uncovers previously unrecognised Foxp3 transcriptional dynamics, establishing a proof-of-concept for integrating CRISPR, Tocky, and machine learning methods as advanced techniques to understand transcriptional dynamics in vivo.

Summary A unique feature of temperate phages is the ability to protect their host bacteria from a second phage infection. Such protection is granted at the lysogenic state, where the phages persist as prophages integrated within the bacterial chromosome, expressing genes that defend the host and themselves from predation. Here, we report a prophage-encoded anti-phage defense system that inhibits DNA packaging of invading phages in Listeria monocytogenes . This system includes a defense protein, TerI, and two self-immunity proteins, anti-TerI1 and anti-TerI2. TerI targets the terminase complex of invading phages to prevent DNA translocation into procapsids without halting the lytic cycle, leading to the release of unpacked non-infectious procapsids upon bacterial lysis. In contrast, the self-immunity proteins, anti-TerI1 and anti-TerI2, counteract TerI during prophage induction to allow virion production. This unique prophage-encoded anti-phage defense system, TERi, is prevalent in Listeria phages, providing population-level host protection without compromising the prophage lytic lifecycle.

Despite the large variety of insect species with divergent morphological, developmental and physiological features questions on gene function could for a long time only be addressed in few model species. The adoption of the bacterial CRISPR-Cas system for genome editing in eukaryotic cells widened the scope of the field of functional genetics: for the first time the creation of heritable genetic changes had become possible in a very broad range of organisms. Since then, targeted genome editing using the CRISPR-Cas technology has greatly increased the possibilities for genetic manipulation in non-model insects where molecular genetic tools were little established. The technology allows for site-specific mutagenesis and germline transformation. Importantly, it can be used for the generation of gene knock-outs, and for the knock-in of transgenes and generation of gene-reporter fusions. CRISPR-Cas induced genome editing can thus be applied to address questions in basic research in various insect species and other study organisms. Notably, it can also be used in applied insect biotechnology to design new pest and vector control strategies such as gene drives and precision guided Sterile Insect Technique. However, establishing CRISPR in a new model requires several practical considerations that depend on the scientific questions and on the characteristics of the respective study organism. Therefore, this review is intended to give a literature overview on different CRISPR-Cas9 based methods that have already been established in diverse insects. After discussing some required pre-conditions of the study organism, we provide a guide through experimental considerations when planning to conduct CRISPR-Cas9 genome editing, such as the design and delivery of guide RNAs, and of Cas9 endonuclease. We discuss the use of different repair mechanisms including homology directed repair (HDR) for a defined insertion of genetic elements. Furthermore, we describe different molecular methods for genetic screening and the use of visible markers. We focus our review on experimental work in insects, but due to the ubiquitous functionality of the CRISPR-Cas system many considerations are transferable to other non-model organisms.

Background Colorectal cancer (CRC) progression from adenoma to adenocarcinoma is associated with global reduction in 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). DNA hypomethylation continues upon liver metastasis. Here we examine 5hmC changes upon progression to liver metastasis. Results 5hmC is increased in metastatic liver tissue relative to the primary colon tumour and expression of TET2 and TET3 is negatively correlated with risk for metastasis in patients with CRC. Genes associated with increased 5-hydroxymethylcytosine show KEGG enrichment for adherens junctions, cytoskeleton and cell migration around a core cadherin (CDH2) network. Overall, the 5-hydroxymethylcyosine profile in the liver metastasis is similar to normal colon appearing to recover at many loci where it was originally present in normal colon and then spreading to adjacent sites. The underlying sequences at the recover and spread regions are enriched for SALL4, ZNF770, ZNF121 and PAX5 transcription factor binding sites. Finally, we show in a zebrafish migration assay using SW480 CRISPR-engineered TET knockout and rescue cells that reduced TET expression leads to a reduced migration frequency. Conclusion Together these results suggest a biphasic trajectory for 5-hydroxymethyation dynamics that has bearing on potential therapeutic interventions aimed at manipulating 5-hydroxymethylcytosine levels.

Oncogenic KRAS mutations underlie some of the deadliest human cancers. Genetic or pharmacological inactivation of mutant KRAS is not sufficient for long-term control of advanced tumors. Using a conceptual framework of pancreatic ductal adenocarcinoma, we find that CRISPR-mediated ablation of mutant KRAS can terminate tumor progression contingent on the concomitant inactivation of STAT3. STAT3 inactivation is needed to ensure that KRAS-ablated tumor cells lose their malignant identity. Mechanistically, the combined loss of mutant KRAS and STAT3 disrupts a core transcriptional program of cancer cells critical to oncogenic competence. This in turn impairs tumor growth in mice and enhances immune rejection, leading to tumor clearance. We propose that the STAT3 transcriptional program operating in cancer cells enforces their malignant identity, rather than providing classical features of transformation, and shapes cancer persistence following KRAS inactivation. Our findings establish STAT3 as a critical enforcer of oncogenic identity in KRAS-ablated tumors, revealing a key vulnerability that could be exploited for combination therapies. Significance The limited clinical success of KRAS inhibitors points to the need to identify means by which tumor cells maintain stemness and immune evasion. We make an unprecedented finding that the STAT3 transcription factor can sustain tumorigenicity of pancreatic cancer cells following depletion of the KRAS oncogenic driver. The results have important implications for successful therapeutic intervention.

The accumulation of protein aggregates has been causatively linked to the pathogenesis of neurodegenerative diseases. In this study, we have conducted a genome-wide CRISPR-Cas9 screen to identify cellular factors that stimulate the degradation of an aggregation-prone reporter protein. Our findings revealed that genes encoding proteins involved in mitochondrial homeostasis, including the translation factor eIF5A, were highly enriched among suppressors of degradation of an aggregation-prone reporter. Conversely, endoplasmic reticulum (ER)-associated ubiquitin ligases facilitated degradation, indicating opposing roles for these cellular compartments in the clearance of aggregation-prone proteins. Genetic or chemical inhibition of eIF5A led to the dissociation of the aggregation-prone substrate from mitochondria, which was accompanied by enhanced degradation through ER-associated ubiquitination. The presence of an aggregation-prone, amphipathic helix that localized the reporter to mitochondria was crucial for the stimulatory effect of eIF5A inhibition. Additionally, the steady-state levels of α-synuclein, a disease-associated protein containing an amphipathic helix that mislocalizes to mitochondria, were reduced upon eIF5A inhibition. We propose that mitochondria behave as a holdout compartment for aggregation-prone proteins, keeping them out of reach of ubiquitin ligases that target them for proteasomal degradation. Therefore, preventing mitochondrial localization of aggregation-prone proteins may offer a viable therapeutic strategy for reducing their levels in neurodegenerative disorders.

The 19S regulatory particle (RP) associates with the 20S core particle (CP) to form the 26S proteasome, an evolutionarily conserved holoenzyme that plays key roles in both physiological and pathological processes. Proteasome inhibitors that target the catalytic subunits within the 20S have proven to be valuable research tools and therapeutics for various cancers. Herein we report the discovery of rapaprotin, a 26S proteasome assembly inhibitor from our natural product-inspired hybrid macrocycle rapafucin library. Rapaprotin induces apoptosis in both myeloma and leukemia cell lines. Genome-wide CRISPR-Cas9 screen identified a cytosolic enzyme, prolyl endopeptidase (PREP) that is required for the pro-apoptotic activity of rapaprotin. Further mechanistic studies revealed that rapaprotin acts as a molecular transformer, changing from an inactive cyclic form into an active linear form, rapaprotin-L, upon PREP cleavage, to block 26S proteasome activity. Time-resolved cryogenic electron microscopy (cryo-EM) revealed that rapaprotin-L induces dissociation of the 19S RP from the 26S holoenzyme, which was verified in cells. Furthermore, rapaprotin exhibits a marked synergistic effect with FDA-approved proteasome inhibitors and resensitizes drug-resistant multiple myeloma cells from patients to bortezomib. Taken together, these results suggest that rapaprotin is a new chemical tool to probe the dynamics of the 26S proteasome assembly and a promising anticancer drug lead.

Background and aim Developmental disorders caused by activating mutations in the RAS-MAPK pathway account for nearly 20% of hypertrophic cardiomyopathy (HCM) cases in paediatric patients. Compared to sarcomeric HCM, RAS-HCM presents a higher risk of obstruction and hospitalisation. The myosin inhibitor mavacamten has been approved in the European Union for treating adults with obstructive HCM; however, clinical trials have excluded syndromic HCM. Consequently, this study aimed to characterise the functional and energetic disturbances induced by the RASopathy mutation BRAF p.Thr599Arg in cardiomyocytes and to evaluate the effects of mavacamten treatment. Methods Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a CRISPR-induced BRAF T599R mutation and their isogenic control were employed to model RAS-HCM in vitro. The cell size, contractility, and transcriptomic profile were assessed to determine the phenotype of the cardiomyocytes. Energetics were evaluated using the Mito Stress assay, live ATP levels, and NAD(P)H and FAD+ autofluorescence. Results BRAF-mutant cardiomyocytes demonstrated hypertrophy and hypercontractility. Furthermore, energetic profiling revealed increased mitochondrial NAD(P)H and FAD+ pools and an enhanced energetic state in the Mito Stress assay with increased maximal respiratory capacity. However, they also exhibited a significant ATP drop during rapid pacing compared to the control, suggesting that mitochondrial capacity remains insufficient to meet the ATP demand. Mavacamten treatment normalised excessive ATP consumption during acute pacing, suggesting reduced mitochondrial overactivity. Conclusions BRAF-mutant cardiomyocytes recapitulate the characteristics of HCM in vitro. Mavacamten mitigates dysfunctions and restores energetic balance under stress conditions, indicating it holds potential as a therapeutic option for RASopathy-associated HCM.

Somatic alterations, like mutations and copy number changes, driver oncogenesis and cancer progression. Their inhibition has been exploited in the clinic, with several targeted therapies approved for patients with specific mutations or amplifications. However, the response rate of these treatments remains low. The causes are several, ranging from clonal heterogeneity to off target binding. For this reason, CRISPR assays have been developed to study the exact effect of a gene’s deletion. Still, the results from them are puzzling with the same alterations responding different to knockout even in the same cellular context. For this reason, we have developed SAEG, a novel deep learning architecture for somatic alterations in cancer. Our architecture is able to model mutations and copy number alterations and protein-protein interactions to predict if a cell will be susceptible to a gene knockout. SAEG outperforms other models and we show that it learns patterns that can be traced back to the biochemical and biological properties of genes and amino acids. Code Availability https://github.com/Luisiglm/SAEG Contact luis.iglesiarmatinez@ucd.ie

Human genome sequencing efforts in healthy and diseased individuals continue to identify a broad spectrum of genetic variants associated with predisposition, progression, and therapeutic outcomes for diseases like cancer 1–6 . Insights derived from these studies have significant potential to guide clinical diagnoses and treatment decisions; however, the relative importance and functional impact of most genetic variants remain poorly understood. Precision genome editing technologies like base and prime editing can be used to systematically engineer and interrogate diverse types of endogenous genetic variants in their native context 7–9 . We and others have recently developed and applied scalable sensor-based screening approaches to engineer and measure the phenotypes produced by thousands of endogenous mutations in vitro 10–12 . However, the impact of most genetic variants in the physiological in vivo setting, including contextual differences depending on the tissue or microenvironment, remains unexplored. Here, we integrate new cross-species base editing sensor libraries with syngeneic cancer mouse models to develop a multiplexed in vivo platform for systematic functional analysis of endogenous genetic variants in primary and disseminated malignancies. We used this platform to screen 13,840 guide RNAs designed to engineer 7,783 human cancer-associated mutations mapping to 489 endogenous protein-coding genes, allowing us to construct a rich compendium of putative functional interactions between genes, mutations, and physiological contexts. Our findings suggest that the physiological in vivo environment and cellular organotropism are important contextual determinants of specific gene-variant phenotypes. We also show that many mutations and their in vivo effects fail to be detected with standard CRISPR-Cas9 nuclease approaches and often produce discordant phenotypes, potentially due to site-specific amino acid selection- or separation-of-function mechanisms. This versatile platform could be deployed to investigate how genetic variation impacts diverse in vivo phenotypes associated with cancer and other genetic diseases, as well as identify new potential therapeutic avenues to treat human disease.

Large scale application of single-cell and spatial omics in models and patient samples has led to the discovery of many novel gene sets, particularly those from an immunotherapeutic context. However, the biological meaning of those gene sets has been interpreted anecdotally through over-representation analysis against canonical annotation databases of limited complexity, granularity, and accuracy. Rich functional descriptions of individual genes in an immunological context exist in the literature but are not semantically summarized to perform gene set analysis. To overcome this limitation, we constructed immune cell knowledge graphs (ICKGs) by integrating over 24,000 published abstracts from recent literature using large language models (LLMs). ICKGs effectively integrate knowledge across individual, peer-reviewed studies, enabling accurate, verifiable graph-based reasoning. We validated the quality of ICKGs using functional omics data obtained independently from cytokine stimulation, CRISPR gene knock-out, and protein-protein interaction experiments. Using ICKGs, we achieved rich, holistic, and accurate annotation of immunological gene sets, including those that were unannotated by existing approaches and those that are in use for clinical applications. We created an interactive website ( https://kchen-lab.github.io/immune-knowledgegraph.github.io/ ) to perform ICKG-based gene set annotations and visualize the supporting rationale.

The Plasmodium falciparum sodium efflux pump Pf ATP4 is a leading antimalarial target, but suffers from a lack of high-resolution structural information needed to identify functionally important features in conserved regions and guide rational design of next generation inhibitors. Here, we determine a 3.7Å cryoEM structure of Pf ATP4 purified from CRISPR-engineered P. falciparum parasites, revealing a previously unknown, apicomplexan-specific binding partner, Pf ABP, which forms a conserved, likely modulatory interaction with Pf ATP4. The discovery of Pf ABP presents a new avenue for designing novel Pf ATP4 inhibitors.

ABSTRACT Double-strand breaks (DSBs) are toxic lesions that lead to genome instability. While canonical DSB repair pathways typically operate independently of RNA, emerging evidence suggests that RNA:DNA hybrids and transcripts near damaged sites can influence repair outcomes. However, a direct role for transcript RNA as a template during DSB repair in human cells is yet to be established. In this study, we designed fluorescent- and sequencing-based assays, which demonstrated that RNA-containing oligonucleotides and messenger RNA serve as templates to promote DSB repair. We conducted a CRISPR/Cas9-based genetic screen to identify factors that promote RNA-templated DSB repair (RT-DSBR), and of the candidate polymerases, we identified DNA polymerase-zeta (Polζ) as the potential reverse transcriptase that facilitates RT-DSBR. Furthermore, by analyzing sequencing data from cancer genomes, we identified the presence of whole intron deletions, a unique genomic scar reflective of RT-DSBR activity generated when spliced mRNA serves as the repair template. These findings highlight RT-DSBR as an alternative pathway for repairing DSBs in transcribed genes, with potential mutagenic consequences.

Background: Gene editing techniques offer new opportunities to improve important traits in aquaculture. The allergenicity of fish flesh is a major problem in aquaculture. Parvalbumin (Parv) is the most prevalent fish allergen. For instance, in salmonids, a single parvalbumin beta-1 protein (parvb1) has been identified as an allergen in specific patients. Therefore, generating trout carrying two parvb1 alleles deleted from the allergenic peptide-encoding region could prevent allergies in these sensitive individuals. Methods Here, we describe the application of the Crispr/cas9 system in an attempt to delete parvb1 exon 2 encoding the allergenic peptide and, alternatively, to replace exon 2 of parvb1 with exon2 of parvalbumin beta-2 protein (parvb2,) which does not encode the allergenic peptide. Exon skipping and swapping were pursued through microhomology-mediated end-joining (MMEJ) knock-In using specifically designed double-stranded donor DNA. Results Genotyping of approximately 200 F0 fingerlings originating from eggs injected with donor DNA designed for exon 2 skipping led to the identification of only one animal carrying an allele lacking exon 2. Genotyping of approximately 150 fingerlings originating from eggs injected with donor DNA for exon 2 swapping did not result in any trout carrying the expected modified allele. Conclusions These preliminary results indicate the potential difficulties associated with the MMEJ KI experiments performed in farmed fish. Finally, new genomic techniques in aquaculture are further discussed in the context of lively debates taking place in the European parliament regarding a possible revision of the current law that determines the legal status of farm animals modified by genome editing. Gene editing, microhomology-mediated end-joining knock-in, parvalbumin, allergenicity, trout, and genetically modified organisms (GMOs).

Loop-extrusion machinery, comprising the cohesin complex and CCCTC-binding factor CTCF, organizes the interphase chromosomes into topologically associating domains (TADs) and loops, but acute depletion of components of this machinery results in variable transcriptional changes in different cell types, highlighting the complex relationship between chromatin organization and gene regulation. Here, we systematically investigated the role of 3D genome architecture in gene regulation in mouse embryonic stem cells under various perturbation conditions. We found that acute depletion of cohesin or CTCF disrupts the formation of TADs, but affects gene regulation in a gene-specific and context-dependent manner. Furthermore, the loop extrusion machinery was dispensable for transcription from most genes in steady state, consistent with prior results, but became critical for a large number of genes during transition of cellular states. Through a genome-wide CRISPR screen, we uncovered multiple factors that can modulate the role of loop extrusion machinery in gene regulation in a gene-specific manner. Among them were the MORF acetyltransferase complex members (Kat6b, Ing5, Brpf1), which could antagonize the transcriptional insulation mediated by CTCF and cohesin complex at developmental genes. Interestingly, inhibition of Kat6b partially rescues the insulator defects in cells lacking the cohesin loader Nipbl, mutations of which are responsible for the developmental disorder Cornelia de Lange syndrome. Taken together, our findings uncovered interplays between the loop extrusion machinery and histone modifying complex that underscore the context-dependent and gene-specific role of the 3D genome.

Summary Genome editing using CRISPR/Cas is a key technology for speeding up breeding for climate-resilient, high-yielding crops (Scheben et al ., 2017). However, efficient targeted mutagenesis requires implementing stable transformation methods and establishing a CRISPR/Cas setup suitable for the species of interest (Shan et al ., 2020). The availability of such methods is a significant bottleneck to advancing many promising, albeit under-researched, crops. Testing and establishing vectors for efficient application of CRISPR/Cas in non-model crops could boost research and breeding of new valuable crops (Ye and Fan, 2021). We edited key pathway genes in the betalain biosynthesis pathway of grain amaranth, i.e., A. hypochondriacus L ., to prove how targeted mutagenesis can be implemented in an orphan crop using the CasCADE modular cloning system (Hoffie, 2022). Grain amaranth is a resilient C 4 dicot orphan crop with excellent nutritional composition. These properties make amaranth a well-suited candidate to be bred as a climate-resilient crop (Joshi et al ., 2018). However, no efficient and reproducible protocol for successful application of CRISPR/Cas9 or stable transformation and regeneration, has been demonstrated in A. hypochondriacus (Castellanos-Arévalo et al ., 2020).

ABSTRACT The axonal membrane-associated periodic skeleton (MPS), consisting of F-actin rings crosslinked by spectrin heterotetramers, is ubiquitous and critical for neuronal function and homoeostasis. However, the initiation and early development of the axonal MPS are poorly understood. Using superresolution imaging, we show that βII-spectrin is recruited early to the axonal cortex, followed by progressive establishment of long-range periodic order. Microtubule dynamics are essential for MPS formation in the early stages, but transition to a passive stabilising role in mature axons. We show that the early subplasmalemmal recruitment of βII-spectrin is dependent on cortical actin but not on actomyosin contractility, and active nucleation of F-actin is required in early development but is dispensable for the mature MPS. Using a βII-spectrin knockout model, we demonstrate that the actin-binding and lipid-interacting domains of βII-spectrin are critical for its subplasmalemmal confinement and, subsequently, MPS maturation. These findings highlight stage-specific cytoskeletal remodelling underlying MPS development and advance our understanding of axonal subcellular architecture.

SUMMARY Wound infection is a major disruptor of wound healing. Keratinocytes, critical in repair and microbial responses, require the L-arginine hydrolysing enzyme arginase1, for effective healing. Wound pathogens such as Pseudomonas aeruginosa may also need L-arginine. We therefore investigated host-microbial interactions in the context of wound healing and L-arginine metabolism. Arginase-inhibited murine wounds challenged with P. aeruginosa, exhibited significantly delayed re-epithelialisation. This finding was recapitulated in vitro using P. aeruginosa- challenged, arginase1 deficient ( shARG1) keratinocytes, associated with reduced epithelial proliferation and viability, and heightened inflammation. Whilst P. aeruginosa challenge promoted host metabolism of L-arginine, this was perturbed in wounded shARG1 keratinocytes. There was, however, heightened downstream polyamine metabolism in shARG1 cells when under P. aeruginosa challenge. Host keratinocyte arginase1 deficiency promoted bacterial growth in vitro , in line with a failure to upregulate the anti-microbial peptides, β-defensins, in shARG1 scratches. This work demonstrates a pivotal role for keratinocyte arginase1 in wound infection. HIGHLIGHTS Host arginase is required for effective healing under P. aeruginosa challenge. P. aeruginosa enhances host keratinocyte L-arginine metabolism upon scratch. P. aeruginosa promotes polyamine metabolism in arginase1 deficient wounds in vitro . Arginase1 is required for keratinocyte anti-microbial defence against P. aeruginosa .

Interferons (IFN) are cytokines that regulate the expression of hundreds of genes during viral infections to generate a broadly antiviral environment in the stimulated cell. Antiviral breadth is provided by the concurrent expression of many individual IFN-stimulated genes (ISG), each encoding a protein with often exquisite antiviral specificity. Here, we show that mechanistic plasticity at a single genetic locus is a novel mechanism to diversify the antiviral profile of human cells. Through alternative splicing, the OAS2 gene encodes two antiviral molecules with distinct target specificities. The shorter OAS2 p69 isoform blocks the replication of seasonal human coronavirus OC43 (HCoV-OC43), while the longer p71 isoform restricts the replication of picornavirus Cardiovirus A (EMCV). The restriction profile is determined by the variable length OAS2 C-terminal tail. Remarkably, the antiviral mechanisms underlying these distinct antiviral profiles are either RNase L dependent or independent, suggesting that splicing divides ‘classic restriction’ versus ‘virus sensing’ systems across two distinct OAS2 polypeptides. Together, our data reveal that the human OAS2 locus uses alternative splicing and mechanistic plasticity to diversify antiviral profiles.