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.