In Atlantic salmon ( Salmo salar ), infectious salmon anemia virus (ISAV) and infectious pancreatic necrosis virus (IPNV) evade host immune response through complex antagonistic mechanisms. Type I interferons (IFNs) play a pivotal role in antiviral defense by signaling through heterodimeric receptors to activate the JAK-STAT pathway and drives the expression of interferon-stimulated genes (ISGs). In this study, CRISPR-Cas9 was used to knock out (KO) interferon receptor genes ( crfb1a , crfb5a , il10rb , ifngr2a ) and a combined group of candidate receptors ( crfb1a , crfb5a , il10rb , ifngr2a , il10r2 ) to investigate their roles and their impact on downstream signaling cascades with RNA sequencing. Recombinant IFNa was used to induce an antiviral state before challenging cells with ISAV and IPNV. The knockouts significantly disrupt downstream antiviral signaling, with two knockouts, crfb1a and crfb5a , showing pronounced effects. During ISAV infection, the crfb1a KO group exhibited a marked reduction in the expression of critical signaling genes such as stat1b, stat2, stat6, and irf3 during ISAV infection, while irf7 was upregulated during IPNV infection. The crfb5a KO group exhibited reduced stat2 expression in ISAV infection and upregulated irf7 during IPNV infection. Despite these disruptions, ISGs such as Mx and isg15 maintained their expression levels across all knockout groups, suggesting potential alternative signaling pathways. Pathway analysis further revealed upregulation of cellular processes like actin regulation and phagosome activity, which may compensate for impaired immune signaling. These findings highlight the distinct roles of IFN receptor genes in mediating antiviral responses and underscore the complexity of IFN signaling in Atlantic salmon.

Background Spatial organization of the genome is fundamental for ensuring accurate gene expression. This process depends on the communication between gene promoters and distal cis -regulatory elements (CREs), which together make up 8% of the human genome and are supported by the chromatin structure. It is estimated that over 90% of disease-associated variants are located in the non-coding region of the genome and may affect CRE. For the cystic fibrosis transmembrane conductance regulator ( CFTR) gene, a complete understanding of tissue-specific CFTR expression and regulation is missing, in particular in the pancreas. Mechanistic insights into tissue-specific expression may provide clarity on the clinical heterogeneity observed in Cystic Fibrosis and CFTR-related disorders. Methods To understand the role of 3D chromatin architecture in establishing tissue-specific expression of the CFTR gene, we mapped chromatin interactions via circular chromosome conformation capture (4C) and epigenomic regulation through H3K27ac and DNase Hypersensitive site I (DHS) in Capan-1 pancreatic cells. Candidate regulatory regions are validated by luciferase reporter assay and CRISPR-knock out. Results We identified active regulatory regions not only around the CFTR gene but also outside the topologically associating domain (TAD). By performing functional assays, we validated our targets and revealed a cooperative effect of the −44 kb, −35 kb, +15.6 kb and 37.7 kb regions, which share common predicted transcription factor (TF) motifs. Comparative 3D genomic analysis and functional assays using the Caco-2 intestinal cell line revealed the presence of tissue-specific CREs. Conclusion By studying the chromatin architecture of the CFTR locus in Capan-1 cells, we demonstrated the involvement of multiple CREs upstream and downstream of the CFTR gene. We also extend our analysis to compare intestinal and pancreatic cells and provide information on the tissue-specificity of CRE. These findings highlight the importance of expanding the search for causative variants beyond the gene coding sequence but also by considering the tissue-specific 3D genome.

ABSTRACT Loss-of-function mutations in ADAMTSL4, a gene encoding an extracellular matrix-associated protein with incompletely understood biological roles, are linked to autosomal recessive disorders predominantly characterized by lens dislocation. Pupil ectopia, increased intraocular pressure, retinal detachment, cataracts, and skeletal abnormalities are also observed in some patients. To investigate ADAMTSL4 biology and related diseases we established a zebrafish knockout line using CRISPR/Cas9 genome editing. The generated zebrafish model harboured the c.234-351del mutation in adamstsl4 , reducing its mRNA levels by 75% in 6 days post fertilization (dpf) larvae, and predicting to produce an inactive protein (p.(Gln78Hisfs*127)). Forty percent of F3 knockout larvae (6 dpf) displayed lethal phenotypes characterized by multiple ocular and non-ocular developmental defects, including pericardial, perivitelline and periocular edema, absence of swim bladder, craniofacial malformations and microphthalmia. The remaining 60% larvae survived and displayed only reduced pupil area, indicating incomplete penetrance of the lethality. Histology revealed extracellular matrix (ECM) and intercellular junctions abnormalities within the cornea, iris, lens, and retinal pigment epithelium (RPE). Adult knockout zebrafish (6 months) presented phenotypes resembling ectopia lentis et pupillae and craniosynostosis, with optical defects in the lens and impaired visual function. ECM and cell junction disorganization in the cornea, lens and RPE were also present in these animals. Transcriptomic analysis revealed disrupted expression of genes involved in development, ECM and cell junctions among other biological processes. These findings show that a damtsl4 recapitulates key features of human ADAMTSL4 -related disorders and that this gene is essential for normal ECM structure, cell junctions and embryonic development. Highlights Zebrafish adamtsl4 knockout recapitulates human ADAMTSL4 -related phenotypes. adamtsl4 is involved in embryonic development. adamtsl4 participates in ECM organization and cell adhesion.

X-linked Lymphoproliferative Syndromes (XLP), which arise from mutations in the SH2D1A or XIAP genes, are characterized by the inability to control Epstein-Barr Virus (EBV) infection. While primary EBV infection triggers severe diseases in each, lymphomas occur at high rates with XLP-1 but not with XLP-2. Why XLP-2 patients are apparently protected from EBV-driven lymphomagenesis, in contrast to all other described congenital conditions that result in heightened susceptibility to EBV, remains a key open question. To gain insights, we cross-compared newly EBV infected versus immune stimulated B-cells from XLP-2 patients or upon XIAP CRISPR knockout, relative to healthy controls. XIAP perturbation impeded outgrowth of newly EBV-infected primary human B-cells, though had no impact on proliferation of B-cells stimulated by CD40 ligand and interleukin-21 or upon established EBV-immortalized lymphoblastoid cell lines (LCLs). B-cells from XLP-2 patients or in which XIAP was depleted by CRISPR editing exhibited a markedly lower EBV transformation efficiency than healthy control B-cells. Mechanistically, nascent EBV infection activated p53-mediated apoptosis signaling, whose effects on transforming B-cell death were counteracted by XIAP. In the absence of XIAP, EBV infection triggered high rates of apoptosis, not seen with CD40L/IL-21 stimulation. Moreover, inflammatory cytokines are present at high levels in XLP-2 patient serum with fulminant EBV infection, which heightened apoptosis induction in newly EBV-infected cells. These findings highlight the crucial role of XIAP in supporting early stages of EBV-driven B-cell immortalization and provide insights into the absence of EBV+ lymphoma in XLP-2 patients. Key points XIAP loss-of-function markedly impairs EBV+ B-cells outgrowth over the first week post-infection, particularly in the presence of IFN-γ. XIAP mutation impedes EBV-driven B-cell transformation by potentiating p53-driven caspase activation and apoptosis.

Rationale WW domain-containing oxidoreductase ( WWOX ) is a gene associated implicated in both neurologic and inflammatory diseases and is susceptible to environmental stressors. We hypothesize partial loss of Wwox function will result in increased sepsis severity and neuroinflammation. Methods Wwox WT/P47T mice, generated by CRISPR/Cas9, and Wwox WT/WT mice were treated with intraperitoneal PBS vs LPS (10mg/kg) and euthanized 12 hours post-injection. Open Field Testing (OFT) and Murine Sepsis Severity Scores (MSS) were utilized to measure sickness behavior and sepsis severity, respectively. Brain tissue was analyzed using immunohistochemistry and PCR to measure neuroinflammation and apoptosis. Results Wwox WT/P47T LPS mice demonstrated a more significant response to sepsis with an increase in sickness behavior, sepsis severity, gliosis, and apoptosis compared to Wwow WT/WT LPS littermates. Conclusions Partial loss of Wwox function increases risk for severe sepsis and neuroinflammation. Given the susceptibility of WWOX to environmental stressors, this may be a target for future therapeutic interventions.

Immune escape is a critical hallmark of cancer progression and underlies resistance to multiple immunotherapies. However, it remains unclear when the genetic events associated with immune escape occur during cancer development. Here, we integrate functional genomics studies of immunomodulatory genes with a tumor evolution reconstruction approach to infer the evolution of immune escape across 38 cancer types from the Pan-Cancer Analysis of Whole Genomes dataset. Different cancers favor mutations in different immunomodulatory pathways. For example, the antigen presentation machinery is highly mutated in colorectal adenocarcinoma, lung squamous cell carcinoma, and chromophobe renal cell carcinoma, and the protein methylation pathway is highly mutated in bladder transitional cell carcinoma and lung adenocarcinoma. We also observe different timing patterns in multiple immunomodulatory pathways. For instance, mutations impacting genes involved in cellular amino acid metabolism were more likely to happen late in pancreatic adenocarcinoma. Mutations in the glucocorticoid receptor regulatory network pathway tended to occur early, while mutations in the TNF pathways were more likely to occur late in B-cell non-Hodgkin lymphoma. Mutations in the NOD1/2 signaling pathway and DNA binding transcription factor activity tended to happen late in breast adenocarcinoma and ovarian adenocarcinoma. Together, these results delineate the evolutionary trajectories of immune escape in different cancer types and highlight opportunities for improved immunotherapy of cancer. Significance Despite its critical role in cancer progression, the evolution of immune escape is poorly understood. We integrate functional genomics and tumor evolution reconstruction and infer immune escape trajectories across cancer types. Our results have important implications for developing biomarkers for immunoprevention and treatment strategies for immune escape of cancer.

Abstract Engineering high-fidelity CRISPR enzymes often leads to reduced cleavage activity, creating a significant hurdle in balancing nuclease specificity and efficiency for clinical applications. Here, we demonstrate that extending the spacer to 21 or 22 nucleotides restores the impaired cleavage activity of SuperFi-Cas9, an engineered high-fidelity Cas9 variant with seven mutations at the PAM-distal region. Structural and mutational analyses reveal that the spacer extension strengthens additional interactions at the PAM-distal end, stabilizing the nuclease– sgRNA–DNA complex, which appears to be disturbed due to the seven mutations. This approach not only provides a high-fidelity Cas9 with uncompromised efficiency but also introduces a novel strategy to enhance CRISPR complex stability. Our findings offer a promising avenue for precise and efficient genome editing, crucial for advancing CRISPR technologies toward clinical translation.

Clearance of incomplete nascent polypeptides resulting from ribosomal stalling is essential for protein homeostasis. While ribosome-associated quality control (RQC) mechanisms that degrade these polypeptides are well-characterized in the cytosol, how stalled endoplasmic reticulum (ER)-bound ribosomes are cleared remains poorly understood. Stalled ER-bound ribosomes are marked by ubiquitin-fold modifier 1 (UFM1) on large ribosomal subunit protein RPL26, but the precise function and regulation of this process are unclear. Here, we demonstrate that canonical RQC factors associate with ribosomes stalled at the ER. Functional cellular assays using ER-targeted stalling reporters reveal that while ribosome splitting is a prerequisite for UFMylation of RPL26, the UFMylation persists without late RQC components that are involved in the clearance of arrested nascent chains (NEMF and LTN1). The UFM1 E3 ligase complex binds to and UFMylates the 60S-peptidyl-tRNA complex and, in concert with the canonical RQC pathway, facilitates the clearance of arrested polypeptides. Our findings reveal that UFMylation acts to maintain translational integrity at the ER.

The Sarm1 NAD + hydrolase drives neurodegeneration in many contexts, but how Sarm1 activity is regulated remains poorly defined. Using CRISPR/Cas9 screening, we found loss of VHL suppressed Sarm1-mediated cellular degeneration. VHL normally promotes O 2 -dependent constitutive ubiquitination and degradation of hypoxia-inducible factor 1 (HIF-1), but during hypoxia, HIF-1 is stabilized and regulates gene expression. We observed neuroprotection after depletion of VHL or other factors required for HIF-1 degradation, and expression of a non-ubiquitinated HIF-1 variant led to even stronger blockade of axon degeneration in mammals and Drosophila . Neuroprotection required HIF-1 DNA binding, prolonged expression, and resulted in broad gene expression changes. Unexpectedly, stabilized HIF-1 prevented the precipitous NAD + loss driven by Sarm1 activation in neurons, despite NAD + hydrolase activity being intrinsic to the Sarm1 TIR domain. Our work argues hypoxia inhibits Sarm1 activity through HIF-1 driven transcriptional changes, rendering neurons less sensitive to Sarm1-mediated neurodegeneration when in a hypoxic state. Competing interests Marc Freeman is co-founder of Nura Bio, a biotech startup pursuing novel neuroprotective therapies including SARM1 inhibition. The remaining authors declare no competing interests.

Long interspersed nuclear element 1 (L1) retrotransposons represent a vast source of divergent genetic information. However, mechanistic analysis of whether and how L1s contribute to human developmental programs is lacking, in part due to the challenges associated with specific profiling and manipulation of human L1 expression. Here we show that thousands of hominoid-specific L1 integrants are expressed in human induced pluripotent stem cells and cerebral organoids. The activity of individual L1 promoters is surprisingly divergent and correlates with an active epigenetic state. Efficient on-target CRISPRi silencing of L1s revealed nearly a hundred co-opted L1-derived chimeric transcripts and L1 silencing resulted in changes in neural differentiation programs and reduced cerebral organoid size. Together, these data implicate L1s and L1-derived transcripts in hominoid-specific CNS developmental processes.

ABSTRACT Chromatin organization is a pivotal factor in stem cell pluripotency and differentiation. However, the role of enhancer looping protein LDB1 in stem cells has not been explored. We generated Ldb1(−/−) embryonic stem cells (ESC) using CRISPR/Cas9 editing and observed a reduction in key stem cell factors SOX2 and KLF4 upon LDB1 loss. Embryoid bodies (EB) derived from Ldb1(−/−) ESC displayed reduced expression of lineage-specific markers and impaired ability to undergo terminal differentiation to erythroblasts. Differential gene expression, including of the Lin28 -mediated self-renewal pathway genes, was observed between WT and Ldb1(−/−) ESC and EB but was most pronounced after differentiation to erythroblasts. LDB1 occupied super enhancers, including those of pluripotency genes, in ESC together with pluripotency factors. LDB1 loss resulted in globally decreased chromatin accessibility in ESC and EB. Conditional LDB1-deficient mice displayed reduced hematopoietic stem cell markers on bone marrow cells, and dysregulation of the Lin28 pathway. Thus, LDB1 function is critical for ESC and EB development and becomes progressively more important during differentiation to erythroblasts.

Abstract Lung cancer, the leading cause of cancer-related mortality, presents major challenges for both standard-of-care therapies and innovative treatments like CAR T cells due to tumor heterogeneity and resistance. To aid in personalized treatment decisions, preclinical models that incorporate these patient-specific factors are essential. In this study, we developed a platform using matched lung tumoroids and healthy lung organoids. The tumoroids retained the identity of the original tumors, as confirmed by genomic, epigenomic, histological, and proteomic analyses. These tumoroids accurately replicated individual patient responses to standard-of-care therapies, highlighting their predictive value. Our platform enabled the identification of individual responses to CAR T cells and associated mechanisms, indicating that efficacy may be influenced by immunoregulatory factors such as autophagy, IL-8 secretion, and PDL1 overexpression. Our modeling strategy shows promise for guiding patient-specific treatment decisions and may facilitate the preselection of lung cancer patients for CAR T-cell therapy.

Abstract The development of novel-acting antidepressant medications with fewer side effects and sustained efficacy requires an in-depth understanding of the aetiology of major depressive disorder (MDD) across diverse populations. Here we used a Mendelian randomization (MR) framework to identify protein levels that influence MDD risk, and that respond to MDD liability in the general population. We use summary-level data from four major ancestral groups to evaluate the consistency of genetic associations and MR estimates across populations. We identified 17 proteins that are putatively causal for MDD, with evidence of differential effects across ancestries for five proteins, which we replicate in independent individual level data. We also identified widespread protein level changes in response to disease liability in the general population. We showed that such associations can appear ancestry-specific until differential power is accounted for, after which the vast majority of associations appear consistent across ancestral groups. The protein response to disease liability can be used to generate a proteomic risk score that is strongly predictive of prospective MDD incidence. Our results indicate that multi-ancestry Mendelian randomization improves power for ancestral groups with smaller sample sizes and will inform our understanding of disease aetiology if differential marginal effects across populations arising due to gene-environment interactions can be studied.

The defence systems bacteria use to protect themselves from their viruses are mechanistically and genetically diverse. Yet the ecological conditions that predict when defences are selected for remain unclear, as substantial variation in defence prevalence has been reported. Experimental work in simple communities suggests ecological factors can determine when specific defence systems are most beneficial, but applying these findings to complex communities has been challenging. Here, we use a comprehensive and environmentally balanced collection of metagenomes to survey the defence landscape across complex microbial communities. We also assess the association between the viral community and the prevalence of defence systems. We identify strong environmental effects in predicting overall defence abundance, with animal-host-associated environments and hot environments harbouring more defences overall. We also find a positive correlation between the density and diversity of viruses in the community and the abundance of defence systems. This study provides insights into the ecological factors that influence the composition and distribution of bacterial defence systems in complex microbial environments and outlines future directions for the study of defence system ecology.

Numerical abnormalities in chromosomal states, referred to as aneuploidy, is commonly observed in many cancer cells. Although numerous internal and external factors induce aneuploidy, the primary cause of aneuploidy in humans remains unclear. DNA damage is identified as a potential cause of aneuploidy by inducing chromosome segregation errors. However, a direct relationship between DNA damage and aneuploidy remains poorly understood. A major reason for this is the extremely low frequency of aneuploidy in cultured cells, making quantitative analyses challenging. In this study, we investigated the relationship between DNA damage and aneuploidy in cell lines containing minichromosomes. These chromosomes are more prone to loss than normal chromosomes, with the rate of loss substantially increased following exposure to various DNA-damaging agents. To determine whether damaged chromosomes were subjected to direct loss or whether chromosome loss occurred as an indirect consequence of a prolonged G2 phase or other factors, we used the CRISPR-Cas9 system to introduce a single DNA double-strand break (DSB) on a minichromosome. The rate of minichromosome loss increased by approximately seven-fold compared with that of the control. Furthermore, the loss rate was significantly elevated in the absence of KU70, a key factor in non-homologous end joining, and upon inhibition of ataxia telangiectasia mutated (ATM), a DNA damage checkpoint protein. Finally, two closely spaced nicks, believed to generate a 5’-overhang, were also shown to induce minichromosome loss. These findings indicated that a single DSB or two closely spaced nicks can cause aneuploidy if improperly repaired in vertebrates.

ABSTRACT RNA-binding proteins (RBPs) are major effectors of post-transcriptional regulation. Recently, we described the role of MEX3A in maintaining intestinal stem cell identity and epithelial renewal by repressing the PPARγ pathway. This work aimed to study MEX3A functional impact in colorectal cancer (CRC). MEX3A and PPARγ expression profiles were characterized in murine and human models. CRISPR/Cas9-mediated MEX3A knockout was performed in patient-derived CRC tumoroids (PDCTs) and MEX3A RNA targets identified through the HyperTRIBE technique. Apc +/fl ; Mex3a +/− mice presented a significant reduction in tumor burden. Apc +/fl ; Kras +/G12D ; Mex3a +/− mice presented a reduced tumor area, while corresponding tumoroids exhibited reduced growth and enhanced differentiation potential mediated by PPARγ signalling. MEX3A overexpression (85% of human CRC cases) was inversely correlated with PPARγ downregulation (72% of cases). Accordingly, MEX3A-depleted PDCTs showed decreased LGR5 expression, accompanied by increased PPARγ expression and higher sensitivity to 5-Fluorouracil/Oxaliplatin (FOLFOX)-based chemotherapy. The HyperTRIBE results revealed a direct interaction between MEX3A and PPARG transcripts. STATEMENT OF SIGNIFICANCE These results emphasize that MEX3A plays a crucial role in colorectal carcinogenesis, partially through regulation of the PPARG pathway, mediating tumour development and response to therapy, thus constituting a potential therapeutic target.

Zebrafish models of genetic epilepsy benefit from the ability to assess disease-relevant knock-out alleles with numerous tools, including genetically encoded calcium indicators (GECIs) and hypopigmentation alleles to improve visualization. However, there may be unintended effects of these manipulations on the phenotypes under investigation. There is also debate regarding the use of stable loss-of-function (LoF) alleles in zebrafish, due to genetic compensation (GC). In the present study, we applied a method for combined movement and calcium fluorescence profiling to the study of a zebrafish model of SCN1A , the main gene associated with Dravet syndrome, which encodes the voltage-gated sodium channel alpha1 subunit (Nav1.1). We evaluated for spontaneous and proconvulsant-induced seizure-like activity associated with scn1lab LoF mutations in larval zebrafish expressing a neuronally-driven GECI (elavl3:GCaMP6s) and a nacre mutation causing a common pigmentation defect. In parallel studies of stable scn1lab s 552 mutant s and F0 crispant larvae generated using a CRISPR/Cas9 multi-sgRNA approach, we find that neither stable nor acute F0 larvae recapitulate the previously reported seizure-like rapid swimming phenotype nor does either group show spontaneous calcium events meeting criteria for seizure-like activity based on a logistic classifier trained on movement and fluorescence features of proconvulsant-induced seizures. This constitutes two independent lines of evidence for a suppressive effect against the scn1lab phenotype, possibly due to the GCaMP6s-derived genetic background (AB) or nacre hypopigmentation. In response to the proconvulsant pentylenetetrazole (PTZ), we see evidence of a separate suppressive effect affecting all conspecific larvae derived from the stable scn1lab s 552 line, independent of genotype, possibly related to a maternal effect of scn1lab LoF in mutant parents or the residual TL background. Nonetheless, both stable and F0 crispant fish show enhanced sensitivity to PTZ relative to conspecific larvae, suggesting that proconvulsant sensitivity provides a more robust readout of scn1lab LoF under our experimental conditions. Our study underscores the unexpected challenges associated with the combination of common zebrafish tools with disease alleles in the phenotyping of zebrafish models of genetic epilepsy. Our work further highlights the advantages of using F0 crispants and the evaluation of proconvulsant sensitivity as complementary approaches that faithfully reflect the shared gene-specific pathophysiology underlying spontaneous seizures in stable mutant lines. Future work to understand the molecular mechanisms by which scn1lab -related seizures and PTZ-related hyperexcitability are suppressed under these conditions may shed light on factors contributing to variability in preclinical models of epilepsy more generally and may identify genetic modifiers relevant to Dravet syndrome.

ABSTRACT D e h ydro d olichyl d iphosphate s ynthase (DHDDS) is an essential enzyme required for several forms of protein glycosylation in all eukaryotic cells. Surprisingly, three mutant alleles, ( Dhdds K42E/K42E (K42E/K42E), Dhdds T206A/K42E (T206A/K42E), and found in only one patient, Dhdds R98W/K42E (R98W/K42E) have been reported that cause non-syndromic retinitis pigmentosa (RP59), an inherited retinal degeneration (IRD). Because T206A was only observed heterozygously with the K42E allele in RP59 patients, we used CRISPR/CAS9 technology to generate T206A/T206A, and subsequently T206A/K42E alleles in mice to assess the contribution of the T206A allele to the disease phenotype, to model the human disease, and to compare resulting phenotypes to our homozygous K42E mouse model. By postnatal (PN) 12-mo, T206A/K42E mice exhibit significant reduction of inner nuclear layer thickness as was observed in K42E/K42E mice. No change in outer nuclear layer thickness is observed in all mutant phenotypes up to PN 12 mo. Electroretinography (ERG) showed a significantly reduced b-wave without a-wave decrement and by PN 3-mo, ERG c- and d-wave responses were significantly attenuated in all phenotypes. Consistent with a reduction in inner nuclear layer thickness seen by OCT and cell loss observed by histology, bipolar and amacrine cell densities were reduced in all Dhdds mutant phenotypes compared to PN 8-12 mo age-matched controls. These results indicate that the DHDDS T206A allele causes retinal disease independent of the K42E allele, and that there likely is a common disease mechanism involving RP59-associated DHDDS mutations. We conclude that the physiological basis of retinal dysfunction in RP59 involves defective signaling in the inner retina resulting in bipolar/amacrine cell degeneration.

Abstract Numerous chemicals are associated with carcinogenesis through epigenetic alterations in cells. To detect global epigenetic changes induced by carcinogens, the housekeeping gene can serve as a reporter locus, offering a baseline for identifying shifts in epigenetic marks. To investigate this potential, we developed a simple, cost-effective, and quantitative reporter system to assess chemically induced epigenetic effects, utilizing the thymidine kinase ( TK ) gene mutation assay as a foundation. Using a standard genotoxicity test cell line, human lymphoblast TK6, we edited the CpG promoter loci of the endogenous TK gene using the CRISPR/dCas9-SunTag-DNMT3A system. This epi-genotoxicity assay, employing modified mTK6 cells, provides a simple method for quantifying chemically induced epigenetic effects. The assay successfully detects both increased TK reversion rates induced by DNMT inhibitors, such as 5-Aza-2'-deoxycytidine and GSK-3484862, and, for the first time, a significant reduction in TK revertant frequency caused by the non-genotoxic carcinogen 12-O-tetradecanoylphorbol-13-acetate (TPA). Chromatin immunoprecipitation and western blotting analyses revealed that TPA treatment led to a global decrease in H3K27Ac levels, likely driven by TPA-mediated inflammation. These results demonstrate the utility of the epi-genotoxicity assay as a valuable tool for evaluating dual-directional epigenetic changes triggered by chemical exposure.

The human genome contains regulatory DNA elements, enhancers, that can activate gene transcription over long chromosomal distances. Here, we show that enhancer distance can be critical for gene silencing. We demonstrate that linear recruitment of the normally distal HBB super-enhancer to developmentally silenced HBG promoters, through deletion or inversion of intervening DNA sequences, results in strongly reactivated HBG expression in adult erythroid cells and ex vivo differentiated hematopoietic stem and progenitor cells. A similar observation is made in the HBA locus, where deletion-to-recruit of the distal enhancer strongly reactivates embryonic HBZ expression. Overall, our work assigns function to seemingly non-regulatory genomic segments: by providing linear separation they may support genes to autonomously control their transcriptional response to distal enhancers.

CRISPR-Cas systems can provide adaptive, heritable immunity to their prokaryotic hosts against invading genetic material such as phages. It is clear that the importance of acquiring CRISPR-Cas immunity to anti-phage defence varies across environments, but it is less clear if and how this varies across different phages. To explore this, we created a synthetic, modular version of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa . We used this synthetic system to test CRISPR-Cas interference against a panel of 13 diverse phages using engineered phage-targeting spacers. We observed complete protection against eight of these phages, both lytic and lysogenic and with a range of infectivity profiles. However, for two phages CRISPR-Cas interference was only partially protective in high nutrient conditions, yet completely protective in low nutrient conditions. This work demonstrates that nutrient conditions modulate the strength of CRISPR-Cas immunity and highlights the importance of environmental conditions when screening defence systems for their efficacy against various phages.

Pseudomonas aeruginosa is a highly versatile and resilient pathogen that can infect different tissues and rapidly develop resistance to multiple drugs. Ceftazidime-avibactam (CZA) is an antibiotic often used to treat multidrug resistant infections, however, the knowledge on the CZA resistance mechanisms in P. aeruginosa is limited. Here we performed laboratory evolution of eight clinical isolates of P. aeruginosa exposed to either CZA or meropenem (MEM) in sub-inhibitory concentrations, and used multi-omics profiling to investigate emerging resistance mechanisms. The majority of strains exposed to MEM developed high resistance (83%, 20/24 strains from eight clinical isolates), with only 17% (4/24) acquiring cross-resistance to CZA. The rate of resistance evolution to CZA was substantially lower (21%, 5/24), while 38% (9/24) acquired cross-resistance to MEM. Whole-genome sequencing revealed strain heterogeneity and different evolutionary paths, with three genes mutated in three or more strains: dacB in CZA-treated strains and oprD and ftsI in MEM-treated strains. Transcriptomic and proteomic analysis underlined heterogeneous strain response to antibiotic treatment with few commonly regulated genes and proteins. To identify genes potentially associated with antibiotic resistance, we built a machine learning model that could separate CZA- and MEM-resistant from sensitive strains based on gene expression and protein abundances. To test some of the identified associations, we performed CRISPR/Cas9 genome editing that demonstrated that mutations in dacB, ampD, and to a lesser extent in mexR directly affected CZA resistance. Overall, this study provides novel insights into the strain-specific molecular mechanisms regulating CZA resistance in Pseudomonas aeruginosa . Importance Pseudomonas aeruginosa is one of the most difficult-to-treat pathogens in the hospital, which often acquires resistance to multiple antibiotics. Ceftazidime-avibactam (CZA) is an essential antibiotic used to treat multidrug resistant infections, but its resistance mechanisms are not well understood. Here we investigated the evolution of resistance to CZA and meropenem (MEM) in eight clinical bacterial isolates from patients’ blood, urine, and sputum. While the rate of resistance evolution to MEM was higher than to CZA, MEM-resistant strains rarely acquired cross-resistance towards CZA. To identify changes at the genome, transcriptome and proteome levels during antibiotic exposure, we performed multi-omics profiling of the evolved strains, and confirmed the effect of several genes on antibiotic resistance with genetic engineering. Altogether, our study provides insights into the molecular response of P. aeruginosa to CZA and MEM and informs therapeutic interventions, suggesting that CZA could still be effective for patients infected with MEM-resistant pathogens.

ABSTRACT Ripening Inducing Factor (RIF) is a key NAC transcription factor regulating strawberry fruit ripening. Previous studies using RIF- RNAi and overexpression lines in Fragaria × ananassa and CRISPR knock-out lines in F. vesca have established the role of RIF in controlling ABA biosynthesis and signaling, cell wall remodeling, and secondary metabolism. In this study, we deciphered FaRIF’s transcriptional regulatory network by combining ChIP-seq-based identification of its direct targets with an analysis of FaRIF -RNAi transcriptome data. These analyses revealed FaRIF’s direct role in multiple aspects of strawberry fruit ripening, including the regulation of ripening-related transcription factors, phytohormone content and signaling, primary and secondary metabolism, and cell wall degradation. Additionally, using the TurboID-based proximity labeling approach, we have identified FaRIF interactors, including proteins involved in mRNA and protein homeostasis, as well as several NAC transcription factors. Among these, FaNAC034 was found to synergistically enhance FaRIF’s transcriptional activity. This integrative analysis, combining transcriptome analysis, in vivo ChIP-seq, and proximity labeling, broadens our knowledge of FaRIF-mediated transcriptional networks and interaction partners, providing valuable insights into the molecular mechanisms underlying strawberry fruit ripening regulation by this transcription factor.

Protoplast-based systems have been utilised in a wide variety of plant species to enable genome editing without chromosomal introgression of foreign DNA into plant genomes. This allows elite cultivars to be edited without further genetic segregation, preserving their unique genetic composition and their regulatory status as non-transgenic. This can be achieved by DNA-free genome editing in protoplasts, followed by regeneration. However, protoplast isolation presents a barrier to the development of advanced breeding technologies in raspberry and no protocol has been published for DNA-free genome editing in the species. Pre-assembled ribonucleoprotein complexes (RNPs) do not require cellular processing and the commercial availability of Cas9 proteins and synthetic guide RNAs has streamlined genome editing protocols. This study presents a novel high-yielding protoplast isolation protocol from raspberry stem cultures and RNP-mediated transfection of protoplast with CRISPR-Cas9. Targeted mutagenesis of the phytoene desaturase gene at two intragenic loci resulted in an editing efficiency of 19%, though estimated efficiency varied depending on the indel analysis technique. Only amplicon sequencing was sensitive enough to confirm genome editing in a low efficiency sample. To our knowledge, this study constitutes the first use of DNA-free genome editing in raspberry. This protocol provides a valuable platform for understanding gene function and facilitates the development of precision breeding in this important soft fruit crop.

Background and aim Mutations in the splice regulator RBM20 account for ∼3 % of genetic cardiomyopathies. In particular, the highly conserved RS domain is a hotspot for disease-associated mutations. Previously, mutations at same amino acid position 634 in the hotspot RS-domain were found to cause dilated cardiomyopathy (DCM) with left ventricular non-compaction (R634L) or without (R634W), but the pathophysiological mechanisms that govern the heterogeneity in phenotype presentation remained unknown. Here, we identify the molecular events caused by the distinct RBM20 mutations from DCM and left-ventricular non-compaction (LVNC) using patient-specific stem cell models. Methods We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) of one LVNC- and two DCM-patients harboring the RBM20-mutations R634L (LVNC) or R634W (DCM). We investigated alternative splicing activity, RBM20 localization, sarcomeric regularity, cAMP level, kinase-specific phosphorylation of key Ca 2+ handling enzymes, physiological cardiac functions as Ca 2+ homeostasis, and metabolic activity on a patient-specific cardiomyocyte level. Force generation was analyzed in patient-specific engineered myocardial tissues. Isogenic rescue and mutation insertion lines were generated by CRISPR/Cas9 technology to analyze the direct impact of the RBM20 mutations on the cardiac phenotype. Results We observed common splicing aberrations for LVNC- and DCM-CM in TTN and RYR2 , RBM20 cytoplasmatic accumulation and irregular sarcomeric structure. LVNC-CM harboring the RBM20-p.R634L variant show distinct molecular, cellular and functional impairments that manifest in CAMK2D , TRDN and IMMT mis-splicing. Splicing defects in LVNC-CM correlate with elevated systolic Ca 2+ and faster Ca 2+ kinetics with elevated cAMP levels and PLN-hyperphosphorylation. An increased metabolic activity and mitochondrial membrane potential support the ‘hyperactive’ LVNC-CM. By contrast, DCM-CM (RBM20-p.R634W) distinctly present with decreased systolic Ca 2+ and increased SR Ca 2+ leak but unchanged Ca 2+ kinetics and metabolic activity. Both mutations lead to severely reduced force of contraction in engineered myocardium. CRISPR/Cas9 gene-edited isogenic control lines of both described RBM20 mutations in LVNC and DCM demonstrated the causative nature of the two mutations and their diverging effects. Further, L-type Ca 2+ channel blockade by verapamil ameliorates the Ca 2+ cycling and leakage phenotypes in LVNC- and DCM-CM. Conclusion We show the first comparative iPSC-model of splice-defect-associated RBM20-dependent LVNC-p.R634L and DCM-p.R634W. We found shared and variant-specific phenotypes on a patient-specific level. Our data demonstrate that the different RBM20 mutations manifest in distinct molecular aberrations in alternative splicing and RBM20 cytoplasmic accumulation that convey various physiological impairments in structure, Ca 2+ handling, metabolism and contractile force.