Development of new and improved tuberculosis (TB) chemotherapies is hampered by antibiotic resistance and drug tolerance by Mycobacterium tuberculosis ( Mtb ). Phenotypic drug tolerance, a phenomenon where Mtb populations can temporarily survive therapeutic antibiotic concentrations, represents a significant hurdle to TB treatment and is indeed one of the factors responsible for prolonged TB therapy. Assays that can identify compounds with improved efficacy against drug tolerant Mtb are urgently required to improve TB treatment regimens. Here, we report the development of a 96-well plate assay capable of identifying anti- Mtb drugs with activity against drug tolerant Mtb in physiologically relevant intracellular environments within macrophages. Primary murine macrophages modified either by immunological activation or specific CRISPR/Cas9 gene knockouts to generate tolerance-inducing environments were infected with an Mtb strain constitutively expressing luciferase. Following drug exposure, differences in bacterial survival were measured by bacterial outgrowth after lysis of the host macrophages. By monitoring Mtb luciferase in infected macrophages before, during and after drug treatment, we confirmed earlier observations that host immune stresses trigger induction of drug tolerance. However, while host stresses induced tolerance against some anti-TB compounds, the same host stresses were synergistic with other anti-TB drugs. Our assay provides the ability to profile the activities of anti-TB drugs on bacteria in intracellular host environments which is critical to the rational design of drug combinations that provide optimal coverage of the Mtb sub-populations in the infected host. Author summary TB treatment is a lengthy process which at minimum takes 6-9 months in cases of drug sensitive Mtb . Long TB treatments are in part, due to drug tolerance phenotypes in the bacterial population which can be a result of immune related stresses on the bacteria in the infected host cells. We used this knowledge to develop a luciferase-based assay which can be used to screen, optimize and discriminate anti-TB drugs with enhanced activity against drug tolerant Mtb inside the infected host cells. Primary murine macrophages were modified either by cytokine activation or genetic knockout of certain host genes to mimic immune related stresses experienced by the bacteria in vivo . By infecting the modified macrophages with an Mtb strain expressing luciferase followed by drug addition, our assay was able to confirm that immune related stress induce drug tolerance to Mtb , and that the drug tolerance phenotypes are unique to certain drug classes consistent with their known mode of action. The assay provides an important addition to anti-TB drug discovery by providing a means to readily screen new drugs and drug combinations that display improved activity in tolerance inducing environments in a physiologically relevant context.

The protein tyrosine phosphatases (PTPs) TCPTP, PTPN22, and SHP1 are critical regulators of the activating phosphotyrosine (pY) site on the initiating T cell kinase, Lck Y394. Still, the broader implications of these phosphatases in T cell receptor (TCR) signalling and T cell biology remain unclear. By combining CRISPR/Cas9 gene editing and mass spectrometry, we evaluate the protein- and pY-level effects of TCPTP, PTPN22, and SHP1 in the Jurkat T cell model system. We find that deletion of each phosphatase corresponds to unique changes in the proteome of T cells, with few large-scale changes to TCR signalling proteins. Notably, PTPN22 and SHP1 deletions have opposing effects on pY abundance globally, while TCPTP deletion modestly elevates pY levels. Finally, we show that TCPTP is indirectly involved in Erk1/2 positive feedback to the TCR. Overall, our work provides evidence for alternative functions of three T cell phosphatases long thought to be redundant.

Summary Melanocytes reside in diverse microenvironments that influence their susceptibility to oncogenic transformation, however, studying rare melanoma subsets has been hindered by the lack of suitable animal models. We developed a primary, immune-competent zebrafish model to study uveal melanoma (UM), utilizing choroidal-targeted injection and electroporation of plasmids containing human GNAQ Q209L and CRISPR/Cas9 cassettes for tumor suppressor gene deletion. Single-cell transcriptional profiling of genetically identical eye- and skin-derived tumors revealed distinct oncogenic pathways, highlighting the importance of studying melanoma subtypes in their correct anatomical context. Additionally, we identified a population of tfec - and pax3a -expressing melanocyte progenitor cells in mitfa -deficient embryos and adult zebrafish eyes, which were highly susceptible to GNAQ-driven transformation. While previous studies have linked mitfa deficiency to accelerated UM onset, our findings suggest that an expanded progenitor population in mitfa -deficient animals drives this susceptibility. Our study establishes a critical role for MITF-independent melanocyte progenitors in UM pathogenesis. Highlights Choroid-targeted injection and electroporation of oncogenic GNAQ induces uveal melanoma in adult zebrafish. Germline loss of mitfa leads to expanded mitfa -independent melanocyte progenitor cells which are highly susceptible to GNAQ-driven transformation. Oncogenic BRAF and GNAQ transform molecularly and developmentally distinct subpopulations of cells within the melanocyte lineage. Abstract Figure Graphical Abstract

Background: /Objectives: Notable similarities in lipid metabolism exist between human and golden Syrian hamster relative to most other rodents. A model for β-thalassemia in hamster was sought via knocking out the hemoglobin β-chain (HBB) gene. There are two HBB genes, as well as seven β-like alleles, predicted in the hamster genome, yet none have been functionally characterized. Methods: To develop a β-thalassemia hamster model and genetically interrogate the functions of the HBB genes in the hamster, we employed CRISPR/Cas9-mediated gene targeting technique and successfully knocked out one of the two hamster HBB genes. Results: Surprisingly, mass spectrometry analysis of the hemolysates from wild type, heterozygous, and homozygous knockout (KO) hamsters showed no changes in the hemoglobin β-chains at protein level. This indicates that the HBB gene that we chose to target does not code for proteins. Interestingly, lipid oxidation during storage was elevated in leg muscle of homozygous KO female hamsters compared to wild type females (P<0.05). Conclusions: Our study provided a path toward developing a hamster β-thalassemia animal model, and related findings suggest an effect of a non-translated HBB gene on oxidative stress. In addition, mass spectrometry provides a way to quickly identify non-protein-coding-genes in species where genomic/transcriptomic annotation is not fully developed.

CRISPR-Cas adaptive immunity systems provide defense against mobile genetic elements and are often countered by diverse anti-CRISPR (Acr) proteins. The Type IE CRISPR-Cas of Escherichia coli K12 has been a model for structural and functional studies and is a part of the species’ core genome. However, this system is transcriptionally silent, which has fueled questions about its true biological function. To clarify the role of this system in defense, we carried out a census of Acr proteins found in Enterobacterales and identified AcrIE9 as a potent inhibitor of the E. coli K12 Type IE CRISPR-Cas system. While sharing little sequence identity, AcrIE9 proteins from Pseudomonas and Escherichia both interact with the Cas7 subunit of the Cascade complex, thus preventing its binding to DNA. We further show that AcrIE9 is genetically linked to AcrIE10, forming the most widespread anti-CRISPR cluster in Enterobacterales , and this module often co-occurs with a novel HTH-like protein with unusual architecture.

Rice (Oryza sativa L.) is a vital global crop with a predominant presence in Asia, including Thailand. However, it faces a significant threat from bacterial blight disease (BB), primarily caused by Xanthomonas oryzae pv. oryzae (Xoo). This research aims to provide an insights into the genetic virulence and variation of Xoo strains isolated in Thailand. Our phylogenetic analysis unveils that the 20 Thai strains align with the Asian strains, setting them apart from African and USA strains. Remarkably, the Average Nucleotide Identity (ANI) values, in comparison to the Xanthomonas oryzae type strain 35933 (XO35933), consistently exceed 99%. These strains can be classified into three assigned ribosomal sequence types (rST). Our investigation into the pangenome and the phylogenetic relationships of these 20 Xoo genomes reveals a diverse genetic landscape, with the pangenome comprising 11,872 orthologous gene clusters, of which roughly 30% form the core genome. Notably, all of these genomes exhibit the presence of a CRISPR-Cas I-C array, indicative of their adaptive immune mechanisms. All strains belonged to BXO1 type LPS cassette with high identity. Furthermore, our analysis identifies two distinct types of plasmids, namely, Xanthomonas oryzae pv. oryzicola strain GX01 plasmid pXOCgx01 (A46, A57, A83, A112, D, and E) and the Xanthomonas oryzae strain AH28 plasmid pAH28 (A97). This genomic resource will be valuable for advancing research on surveillance, prevention, management, and comparative studies of this critical pathogen in the future.

Foraging is essential for sustenance and well-being of all organisms. The transition from well-fed to food-deprived conditions in C. elegans triggers a localized exploration of the environment characterized by frequent reorientations. However, over time the cumulative frequency of these reorientations decreases, facilitating the transition to global search behaviour. To investigate the genetic regulation of foraging in C. elegans , we conducted a screen of neuropeptide mutants and identified several candidates involved in modulating this behaviour. Among these, neuropeptide FLP-15 emerged as a key regulator of both local and global search behaviours. Our observations revealed that FLP-15 regulates the frequency and duration of reversals during foraging. Further investigation indicated that FLP-15 is expressed in and functions through the I2 pharyngeal neuron via the G-protein coupled receptor NPR-3. Mutants lacking either flp-15 or npr-3 displayed a significant decrease in reversal frequency during local search behaviours. Interestingly, unlike wild-type animals, the reversal frequency in flp-15 and npr-3 mutants did not decrease over time. This study also describes the expression pattern of NPR-3, in a subset of head neurons, predominantly comprising of dopaminergic neurons. This expression pattern highlights a potential link between neuropeptide signalling and dopaminergic modulation of behaviour. Finally, exogenous dopamine supplementation assays revealed that FLP-15 may regulate foraging by modulating dopamine transmission, highlighting a novel neuropeptide-dopamine interaction involved in the control of foraging behaviours.

ABSTRACT Non-viral gene editing offers a practical alternative to viral delivery for durable biologics production. Clinical trials have shown that adeno-associated virus encoding broadly neutralizing antibodies can protect against HIV, but result in limited, short-lived responses. The development of non-viral gene editing approaches in hematopoietic stem and progenitor cells holds promise for long-term antibody production. In this study, we evaluated CRISPR/Cas9 and CRISPR/Cas12a for gene knock-in at the immunoglobulin heavy chain locus in non-human primate hematopoietic stem and progenitor cells. Delivering the nuclease as a protein alongside a custom DNA template, we optimized editing with Cas12a and demonstrated higher knock-in efficiency and fewer non-specific edits than Cas9. Transplantation of edited non-human primate hematopoietic stem and progenitor cells into MISTRG mice led to engraftment, B cell differentiation, and transgene expression of a reporter transgene or anti-HIV antibody after HIV immunization with detectable anti-HIV antibody titers in peripheral blood circulation. These findings demonstrate the feasibility of using non-viral gene editing in HSPC as a potential strategy for sustained biologics production in the treatment of chronic diseases such as HIV. Future work will assess the efficacy of this model in a non-human primate model of HIV infection.

CRISPR–Cas9 gene editing holds transformative promise for genetic therapies, but is hindered by off-target effects that undermine its precision and safety. To address this, we developed CRISMER, a hybrid deep-learning architecture that uses multi-branch convolutional neural networks to extract k-mer features and transformer blocks to capture long-range dependencies. This hybrid approach enhances the prediction and optimization of single-guide RNA (sgRNA) designs. CRISMER was trained on Change-seq and Site-seq datasets, using a 20 × 16 sparse one-hot encoding scheme, and evaluated on independent datasets including Circle-seq, Guide-seq, Surro-seq, and TTISS. CRISMER outperformed existing tools, achieving an F1 score of 0.7092 and a PR-AUC of 0.8006 on the CRISPR-DIPOff dataset. It also excelled in measuring sgRNA specificity and optimizing designs for genes, such as PCSK9 and BCL11A, yielding sgRNAs with reduced off-target activity. For example, a G-to-C mutation at position 12 in the sgRNA for PCSK9 and at position 11 for BCL11A led to significant improvements in off-target profiles. Interpretability analysis via integrated gradients confirmed the model’s focus on critical PAM-proximal regions and mismatch patterns. These results demonstrate that CRISMER significantly improves the accuracy and safety of CRISPR-Cas9, advancing its reliability for therapeutic applications.

Abstract ​Spatially resolved in vivo CRISPR screening integrates gene editing with spatial transcriptomics to examine how genetic perturbations alter gene expression within native tissue environments. However, current methods are limited to small perturbation panels and the detection of a narrow subset of protein-coding RNAs. We present Perturb-DBiT, a distinct and versatile approach for the simultaneous co-sequencing of spatial total RNA whole-transcriptome and single-guide RNAs (sgRNAs), base-by-base, on the same tissue section. This method enables unbiased discovery of how genetic perturbations influence RNA regulation, cellular dynamics, and tissue architecture in situ. Applying Perturb-DBiT to a human cancer metastatic colonization model, we mapped large panels of sgRNAs across tumor colonies in consecutive tissue sections alongside their corresponding total RNA transcriptomes. This revealed novel insights into how perturbations affect long non-coding RNA (lncRNA) co-variation, microRNA–mRNA interactions, and global and distinct tRNA alterations in amino acid metabolism linked to tumor migration and growth. By integrating transcriptional pseudotime trajectories, we further uncovered the impact of perturbations on clonal dynamics and cooperation. In an immune-competent syngeneic mouse model, Perturb-DBiT enabled investigation of genetic perturbations within the tumor immune microenvironment, revealing distinct and synergistic effects on immune infiltration and suppression. Perturb-DBiT provides a spatially resolved comprehensive view of how genetic knockouts influence diverse molecular and cellular responses including small and large RNA regulation, tumor proliferation, migration, metastasis, and immune interactions, offering a panoramic perspective on perturbation responses in complex tissues.

The evolution of genome engineering technologies has transformed biomedical research, enabling precise and efficient modification of genetic material Doudna and Charpentier, 2014. Among these, CRISPR-Cas9 stands out as a revolutionary gene-editing tool, though it often requires extensive expertise and technical knowledge Cong et al., 2013; J. G. Doench et al., 2016. We propose GeneFix-AI, an Artificial Intelligence (AI)-driven platform for real-time prediction and correction of genetic mutations in non-human species. Developed using cutting-edge models inspired by recent advances at Harvard and Peking University Chen et al., 2021; Wu et al., 2020, GeneFix-AI integrates machine learning to predict mutations, design optimal guide RNAs, and evaluate editing outcomes. This system aims to automate the CRISPR-Cas9 workflow, making high-precision gene editing more accessible to researchers without extensive molecular biology backgrounds Liu et al., 2019. We present the system architecture, training methodology, and potential impact of GeneFix-AI in democratizing genome editing and accelerating discoveries in genetics.

Abstract Background: CRISPR-Cas9 technology is a powerful tool for precise genome editing and is increasingly applied to correct genetic mutations associated with various diseases, including cancer. This system utilizes a single-guide RNA (sgRNA), typically 20 base pairs long and complementary to the target DNA sequence, to direct the Cas9 nuclease for targeted gene activation (knock-in) or repression (knockout). In recent advancements in cancer immunotherapy, CRISPR-Cas9 has been extensively used to enhance the efficacy of Chimeric Antigen Receptor (CAR) T-cell therapy. The development of universal CAR T cells involves the knockout of key genes such as TRAC (T-cell receptor alpha chain), B2M (Beta-2 microglobulin), and PDCD1 (Programmed cell death protein 1), which improves T-cell persistence, immune evasion, and anti-tumor function. Method: In this study, sgRNAs targeting PDCD1, B2M, and TRAC were designed using nine widely recognized AI-driven bioinformatics tools: CHOPCHOP, CRISPOR, GenScript, Benchling, Cas-Designer, E-CRISP, CRISPR-ERA, CRISPRscan, and ATUM gRNA Tool. These platforms use various algorithms and genomic datasets to predict sgRNA candidates with high on-target activity and minimal off-target effects. The selected sgRNAs were assessed based on criteria including GC content, self-complementarity, and exon targeting. Results: The sgRNA design tools consistently identified high-confidence target sites within exon 1 of the PDCD1, TRAC, and B2M genes. For PDCD1 (PD-1), the sgRNA sequence (5′-CACGAAGCTCTCCGATGTGT-3′) was selected as the most optimal candidate, showing strong consensus across all platforms. Similarly, for TRAC, the sgRNA (5′-TCTCTCAGCTGGTACACGGC-3′) targeting exon 1 was chosen based on its high predicted efficiency and specificity. In the case of B2M, the sgRNA (5′-GAGTAGCGCGAGCACAGCTA-3′) was identified as an ideal target site within exon 1, a region critical for MHC class I expression and immune evasion. These sgRNAs demonstrated favorable characteristics including appropriate GC content, minimal self-complementarity, and low predicted off-target activity. To ensure their functional reliability, all selected sgRNAs were validated through an extensive review of scientific literature and previously published patent data, confirming their utility in gene knockout studies related to CAR T-cell enhancement. Conclusion: Among the tools evaluated, CHOPCHOP, Benchling, and CRISPOR emerged as the most comprehensive, offering robust information on GC content, self-complementarity, exon identification, and detailed off-target predictions. Additionally, this study compiled a list of relevant clinical trials involving gene knockouts of PDCD1, TRAC, and B2M to further support the therapeutic relevance of these targets in CAR T-cell development.

Streptococcus mutans is a major cause of dental caries worldwide. Targeted therapeutic strategies to eradicate S. mutans include oral phage rinses. In this study, we investigated how phage resistance develops in S. mutans . As a model phage, we used ɸAPCM01, which is known to infect a serotype e strain. We isolated and sequenced the genomes of 15 spontaneous resistant mutants and found that 10 had acquired novel CRISPR spacers targeting the phage, with a total of 18 new spacers identified. Additionally, eight strains contained mutations in rhamnose-glucose polysaccharide (RGP) biosynthetic genes, three of which also acquired spacers. Only the rgp mutants exhibited defects in phage absorption, supporting the role of these cell surface glycans as the phage receptor. Mutations in rgpF and the newly identified gene rgpX led to severe cell division defects and impaired biofilm formation, the latter of which shared by the rgpD mutant. Thus, rgp mutations confer phage resistance but impose severe fitness costs, limiting pathogenic potential. Surprisingly, we found that ɸAPCM01 was capable of binding to and injecting its genome into UA159, a model serotype c strain. However, UA159 was resistant to infection due to an unknown post-entry defense mechanism. Consequently, ɸAPCM01 has the potential to infect both major serotypes associated with dental caries. Repositories The genome sequence of Streptococcus mutans DPC6143 was deposited at NCBI with the accession number NZ_CP172847.1.

ABSTRACT Neuromodulators such as the monoamines are known to differ from classical neurotransmitters like glutamate in the time scale of signaling due to activation of slower G protein-coupled receptors. Recent work has suggested that the mode of release also differs between classical and modulatory transmitters. Although many components of neurotransmitter release machinery have been identified, we still understand little about the mechanisms responsible for differences in release. In this study, we address the differences between release of dopamine and glutamate by comparing the composition of synaptic vesicles (SVs) that contain the vesicular monoamine transporter 2 (VMAT2) versus vesicular glutamate transporter 2 (VGLUT2). Previous work has shown that these SV populations differ in frequency dependence, recycling kinetics and biogenesis. Taking advantage of a CRISPR-generated knock-in mouse with a cytoplasmic hemagglutinin (HA) tag at the N-terminus of VMAT2 to immunoisolate monoamine SVs, we find differences in the abundance and isoform expression of many SV protein families. Validation in primary neurons and in brain tissue confirms these differences in SV protein abundance between dopamine and glutamate release sites. Functional analysis reveals that the loss of differentially expressed SCAMP5 selectively impairs the recycling of VGLUT2 SVs, sparing VMAT2 vesicles in the same neuronal population. These findings provide new insights into the molecular diversity of SVs and the mechanisms that regulate the release of dopamine and glutamate, with implications for the physiological role of these transmitters and behavior.

ABSTRACT Receptor-interacting protein kinase 2 (RIPK2) has emerged as a promising drug target in various cancers, including prostate cancer (PC). However, the absence of reliable biomarkers to assess RIPK2 activity limits both patient selection for anti-RIPK2 therapies and treatment monitoring. To address this gap, we performed RNA-Seq analysis on PC cell lines (22Rv1, DU145, and PC3) with CRISPR/Cas9-mediated RIPK2 knockout ( RIPK2 -KO) using two independent guide RNAs. This analysis identified 13 candidate RIPK2-regulated genes, of which eight were validated by reverse transcription quantitative PCR (RT-qPCR). Furthermore, treatment with two distinct RIPK2 inhibitors significantly reduced RIPK2 signature scores in five independent PC cell lines in a dose- and/or time-dependent manner. Clinical association analyses revealed that high RIPK2 signature scores correlate with metastasis and worse biochemical recurrence-free, progression-free, disease-free, and overall survival, outperforming RIPK2 mRNA levels as a prognostic biomarker. This study establishes, for the first time, a RIPK2-regulated gene signature as a potential biomarker for RIPK2 activity and PC prognosis, warranting further validation in clinical specimens to provide a much-needed tool for patient stratification and response monitoring in RIPK2-targeted therapies.

Cryptosporidium is an apicomplexan parasite that causes diarrhoeal disease. The species C. parvum is zoonotic and causes significant morbidity and mortality for both humans and farm animals; most commonly, calves and lambs. A One Health approach that integrates human, animal and environmental health perspectives is required to tackle this disease. Current treatments are limited and ineffective, meaning there is an urgent need to develop new anti-cryptosporidials both for human and animal health. The neonatal calf model is a natural model of infection employed as a tool for drug discovery or generating parasite material. However, the model is seldom utilised to investigate host-parasite interaction. Fundamental information about this model, including the location of the parasite in the gut, is lacking. It is also unclear how the more commonly utilised immunocompromised mouse models of cryptosporidiosis compare to the neonatal calf model. To address this, we established an acute, moderate experimental C. parvum infection in neonatal calves. Using transgenic parasites, we created a tissue atlas of infection for neonatal calf gut and immunocompromised mouse models and mapped and quantified infection to draw robust comparisons between models. Cryptosporidium infection was observed at high levels throughout the neonatal calf gastrointestinal tract and was not limited to the ileal-cecal junction, as previously suggested. This infection pattern is most similar to the acute cryptosporidiosis mouse model, interferon-gamma knockout mice (IFNγKO). Infection with transgenic parasites allowed us to perform in vivo and ex vivo tissue imaging of the chronic cryptosporidiosis mouse model, NOD SCID Gamma KO (NSG) mice. In contrast, in NSG mice infection is low in the small intestines and highest in the caecum and colon. Understanding the true distribution of infection in the gastrointestinal tract of these three key animal models provides new perspectives on how to interpret and design drug efficacy studies and provides new insight into host-pathogen interaction.

Abstract CRISPR/Cas9-mediated genome editing is a powerful tool for producing animal models of human diseases. However, it often encounters challenges related to low efficiency of donor DNA templates insertion through homology-directed repair (HDR) pathway or unwanted insertions and/or multiplications. Here, we present findings from multiple targeting experiments aimed at generating a Nup93 conditional knockout (cKO) mouse model. Injection of CRISPR/Cas9 components into over two thousand zygotes, resulted in 270 founder animals. Our study revealed various obstacles associated with the use of single-stranded (ssDNA) and double-stranded DNA (dsDNA) templates during cKO generation, highlighting the critical role of denaturation of long 5’-monophosphorylated dsDNA templates in enhancing precise genome editing and reducing template multiplications. Application of RAD52 protein increased HDR efficiency of ssDNA integration almost 4-fold, albeit with an associated increase in template multiplication. Targeting the antisense strand of DNA using two crRNAs demonstrated better efficacy in HDR-mediated precise genome editing when compared to targeting the sense or sense-antisense strands. In addition, the application of 5’-end biotin-modified donor DNA resulted in up to a 8-fold increase in HDR-mediated single-copy template integration compered to unmodified dsDNA donor. Furthermore, application of 5’-end C3 spacer modified template resulted in up to a 20-fold increase in correctly HDR modified mice independent from ssDNA or dsDNA template employment. This study underscores potential pitfalls in CRISPR/Cas9-mediated genome editing and offers simple practical solutions to refine this potent tool. These findings highlight various strategies to enhance CRISPR/Cas9 HDR efficiency, providing a framework for improving precision in the generation of conditional knockout models.

CRISPR and their associated Cas proteins provide adaptive immunity in prokaryotes, protecting against invading genetic elements. These systems are categorized into types and are highly diverse. Genomes often harbor multiple CRISPR arrays varying in length and distance from Cas loci. However, the ecological roles of multiple CRISPR arrays and their interactions with multiple Cas loci remain poorly understood. We present a comprehensive analysis of CRISPR systems that uncovers variation between diverse Cas types regarding the occurrence of multiple arrays, the distribution of their lengths and positions relative to Cas loci, and the diversity of their repeat sequences. Some types tend to occurr as the sole Cas present, but typically comprise two or more arrays, especially for types I-E and I-F. Multiple Cas types are also common, with some systems showing a preference for specific co-occurrence. Distinct array distributions and orientations around Cas loci indicate substantial differences in functionality and transcriptional behavior among Cas types. Our analysis suggests that arrays with identical repeats in the same genome acquire new spacers at comparable rates, irrespective of their proximity to the Cas locus. Furthermore, repeat similarities in our data set indicate that arrays of systems that often co-occur with other systems tend to have more diverse repeats than those mostly appearing alongside solitary systems within the genome. Our analysis suggests that co-occurring Cas type pairs might not only collaborate in spacer acquisition but also maintain independent and complementary functions and that CRISPR systems distribute their defensive spacer repertoire equally across multiple CRISPR arrays.

Glucocorticoids (GCs) are potent modulators of immune responses; however, the mechanisms by which GCs regulate gene expression in human CD8 T cells remain incompletely defined. Here, we delineate how physiological cortisol signalling shapes the transcriptional and chromatin landscapes of primary human CD8 T cells. We identify a substantial cohort of GC-responsive genes that are co-regulated through the cooperative activity of the glucocorticoid receptor (GR) and RUNX transcription factors. Integrative RNA sequencing and ChIP sequencing analyses identified genome-wide cortisol-responsive immunoregulatory genes. Genetic deletion of the glucocorticoid receptor (GR, encoded by NR3C1 ) abolished cortisol-induced gene expression changes, confirming GR-dependency. Notably, GR chromatin occupancy in cortisol-treated CD8 T cells was strongly enriched at RUNX transcription factor (TF) motifs rather than canonical glucocorticoid response elements (GREs). Co-immunoprecipitation assays validated a ligand-dependent physical interaction between GR and RUNX. Single-cell transcriptomic analyses of tumour infiltrating CD8 T cells revealed significant enrichment of cortisol-responsive genes, indicating an active GC signalling (response) within the tumour microenvironment. GR-RUNX dual controlled genes were enriched in tumour-infiltrating CD8 T cells across multiple cancer types, including lung adenocarcinoma, head and neck squamous carcinoma, pancreatic cancer, and breast cancer. We found GR-RUNX co-regulated genes are mostly expressed in the exhausted CD8 T cell population of different solid tumours These results suggest that local cortisol signalling within tumour microenvironments drives CD8 T cell dysfunction through GR-RUNX TF cooperation. Collectively, our findings identify RUNX TF as a critical mediator of GR signalling in human CD8 T cells and reveal a novel mechanism by which endogenous glucocorticoids influence antitumour immunity, which could be therapeutically targetable.

Proteins of the cytohesin family are known for their guanine-nucleotide exchange factor function for ARF-GTPases, mainly for ARF1 and ARF6. While Arf1 and Arf6 deficiency results in embryonic lethality, in vivo functions of cytohesins are rarely described and mostly inconspicuous. We analyzed the role of cytohesin-2 in vivo and in vitro and found that cytohesin-2 full knockout mice die within one day after birth. Mass spectrometry-based organellar proteomics in wildtype and CRISPR-Cas9-generated cytohesin-2 -/- C2 myoblasts revealed a markedly altered Golgi compartment. Golgi volumes were reduced in different cytohesin-2 -/- cell lines compared to wildtype cells as revealed by immunofluorescence. Reduced Golgi volumes were rescued by introducing cytohesin-2. Finally, we observed that typical functions of the Golgi apparatus were disrupted in cytohesin-2-deficient cells. Cytohesin2 -/- C2 myoblasts exhibited significant changes in the galactose / N-acetyl-galactosamine glycosylation on the cell surface compared to wildtype cells when stained with peanut agglutinin. Further, protein secretion was overall reduced in neonatal cytohesin-2 -/- mice compared to wildtype as determined by mass spectrometry-based proteomics. This study describes the essential role of cytohesin-2 in neonatal development and a novel function of the protein in Golgi regulation.

Sex-specific penetrance in autosomal dominant Mendelian conditions is largely understudied. The neurodevelopmental disorder Pilarowski-Bjornsson syndrome (PILBOS) was initially described in females. Here, we describe the clinical and genetic characteristics of the largest PILBOS cohort to date, showing that both sexes can exhibit PILBOS features, although males are overrepresented. A mouse model carrying a human-derived Chd1 missense variant ( Chd1 R 616 Q /+ ) displays female-restricted phenotypes, including growth deficiency, anxiety and hypotonia. Orchiectomy unmasks a growth deficiency phenotype in male Chd1 R 616 Q /+ mice, while testosterone rescues the phenotype in females, implicating androgens in phenotype modulation. In the gnomAD and UK Biobank databases, rare missense variants in CHD1 are overrepresented in males, supporting a male protective effect. We identify 33 additional highly constrained autosomal genes with missense variant overrepresentation in males. Our results support androgen-regulated sexual dimorphism in PILBOS and open novel avenues to understand the mechanistic basis of sexual dimorphism in other autosomal Mendelian disorders. Graphical Abstract

RNA-binding proteins (RBPs) are important regulators of post-transcriptional gene expression. Understanding which and how RBPs promote cancer progression is crucial for cancers that lack effective targeted therapies such as triple negative breast cancer (TNBC). Here, we employ both in vitro and in vivo pooled CRISPR/Cas9 screening to identify 50 RBP candidates that are essential for TNBC cell survival. Integrated eCLIP and RNA-sequencing analysis identify that poly(U)-binding splicing factor 60 (PUF60) drives exon inclusion within proliferation-associated transcripts that, when mis-spliced, induce cell cycle arrest and DNA damage. Furthermore, disrupting PUF60 interactions with 3’ splice sites via a substitution in its RNA-binding domain causes widespread exon skipping, leading to downregulation of proliferation-associated mRNAs and inducing apoptosis in TNBC cells. We demonstrate that loss of PUF60-RNA interactions inhibits TNBC cell proliferation and shrinks tumor xenografts, revealing the molecular mechanism by which PUF60 supports cancer progression. Significance Our work demonstrates functional in vivo screening of RBPs as an effective strategy for identifying unexpected cancer regulators. Here, we reveal a crucial role for PUF60-mediated splicing activity in supporting oncogenic proliferation rates and highlight its potential as a therapeutic target in triple negative breast cancer.

Genome wide association studies have identified multiple loci that mediate the risk of developing late-onset Alzheimer’s Disease (LOAD). The gene WW-domain containing oxidoreductase ( WWOX ) has been identified in recent LOAD risk meta-analyses, yet its function in the brain is poorly understood. Using Drosophila, we discovered that knockdown of the highly conserved Wwox gene impacts longevity and sleep, having roles in both neuronal and glial subtypes. In an amyloid beta 42 (Aβ 42 ) transgenic model of AD, RNAi-mediated knockdown of Wwox significantly decreased both lifespan and locomotion whilst elevating soluble Aβ 42 . Transcriptomic and metabolomic analyses revealed that these effects were accompanied by elevated lactate dehydrogenase ( Ldh ) mRNA and lactate levels, downstream of an increase in the key unfolded protein response protein Atf4. Strikingly, we found that upregulation of Wwox in the Aβ 42 model through CRISPR activation significantly reduced amyloid load, improved longevity and locomotion. Multi-omics analysis revealed Wwox upregulation partially reversed several key Aβ 42 -induced transcriptional pathways in the brain and reduced levels of L-methionine and associated enzymes. These findings support a role for reduced WWOX levels in the genetic risk of developing LOAD via pyruvate metabolism and point towards WWOX activation as a protective therapeutic strategy.

We combine Lattice Structured Illumination Microscopy ( di SIM or SIM 2 with ∼60 nm resolution), Lattice Light-sheet microscopy and Fluorescence Recovery After Photobleaching (FRAP) to explore 53BP1 dynamics in Retinal Pigment Epithelial cells. 53BP1 forms liquid condensates during double-strand DNA repair, long-range DNA end-joining and heterochromatin maintenance. Our super-resolution movies reveal differences in 53BP1 foci contour: some foci are compact and stationary while others appear amorphous, dynamically changing shapes. To explore them, we developed FRAP in the Super-Resolution regime (FRAP-SR). 53BP1 foci with an amorphous loose contour display subcompartments that recover 53BP1-eGFP signals rapidly, indicating differential protein mobilities and 53BP1 functions within a single foci. In contrast, 53BP1-eGFP foci with a compact contour recover uniformly as single foci but show higher heterogeneity in 53BP1-eGFP recovery rates compared to foci that recover as multiple subcompartments. In cells released from aphidicolin, amorphous foci show faster 53BP1 recovery compared to compact foci. We discuss the conceptual implications of different 53BP1 mobilities, and how the FRAP-SR method transforms studies of dynamic 60-100 nm structures.

ABSTRACT Human induced pluripotent stem cells (iPSCs) offer immense potential as a source for cell therapy in spinal cord injury (SCI) and other diseases. The development of hypoimmunogenic, universal cells that could be transplanted to any recipient without requiring a matching donor, could significantly enhance their therapeutic potential and accelerate clinical translation. To create off-the-shelf hypoimmunogenic cells, we used CRISPR-Cas9 to delete B2M (HLA class I) and CIITA (master regulator of HLA class II). Double-knockout (DKO) iPSC-derived neural progenitor cells (NPCs) evaded T cell-mediated immune rejection in vitro and after grafting into the injured spinal cord of athymic rats and humanized mice. However, loss of HLA class I heightened susceptibility to host natural killer (NK) cell attack, limiting graft survival. To counter this negative effect, we engineered DKO NPCs to overexpress macrophage migration inhibitory factor (MIF), an NK cell checkpoint ligand. MIF expression markedly reduced NK cell-mediated cytotoxicity and improved long-term engraftment and integration of NPCs in the animal models for spinal cord injury. These findings demonstrate that MIF overexpression, combined with concurrent B2M and CIITA deletion, generates hiPSC neural derivatives that escape both T- and NK-cell surveillance. This strategy provides a scalable route to universal donor cells for regenerative therapies in SCI and potentially other disorders.