Human brain development is highly regulated by several spatiotemporal processes, which disruption can result in severe neurological disorders. Emerging evidence highlights the pivotal role of mitochondrial function as one of these fundamental pathways involved in neurodevelopment. Our study investigates the role of 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) protein in cortical neurogenesis and mitochondrial activity, since mutations in the HPDL gene are associated with SPG83, a childhood-onset form of hereditary spastic paraplegia characterized by corticospinal tract degeneration and cortical abnormalities. Starting from mutant neuroblastoma cells, we demonstrated that HPDL is essential to mitochondrial respiratory chain supercomplex assembly and cellular redox balance. Moreover, transcriptomic analyses revealed dysregulated pathways related to neurogenesis, implicating HPDL role in early cortical development. To further elucidate the role of HPDL, we generated cortical neurons and organoids from SPG83 patient-derived induced pluripotent stem cells. Mutant cells exhibited premature neurogenesis at early differentiation stages, likely leading to depletion of cortical progenitors, as evidenced by decreased proliferation, slight increase of apoptosis, and unbalanced cortical type composition at later stages. Furthermore, cortical organoids derived from SPG83 patients showed impaired growth, reminding microcephaly observed in severe cases. In addition, mitochondrial morpho-functional characterization in mutant neurons confirmed disruption of OxPhos chain functionality and increased ROS generation rate. Treatment of cortical cells with two antioxidant compounds, could partially revert premature neurogenesis. In conclusion, our findings reveal a critical role for HPDL in coordinating cortical progenitor proliferation, neurogenesis, and mitochondrial function. These insights shed light on a mechanistical understanding of SPG83 pathology and underscore the therapeutic potential of targeting oxidative stress in this and related neurological disorders.

ABSTRACT Zinc-finger Antiviral Protein (ZAP)-mediated RNA decay (ZMD) restricts replication of viruses containing CpG dinucleotide clusters. However, why ZAP isoforms differ in antiviral activity and how they recruit cofactors to mediate RNA decay is unclear. Therefore, we determined the ordered events of the ZMD pathway. The long ZAP isoform preferentially binds viral RNA, which is promoted by TRIM25. The endoribonuclease KHNYN then cleaves viral RNA at positions of ZAP binding. The 5’ cleavage fragment undergoes TUT4/TUT7-mediated 3’ uridylation and degradation by DIS3L2. The 3’ cleavage fragment is degraded by XRN1. ZAP and TRIM25 interact with KHNYN, TUT7, DIS3L2 and XRN1 in a RNase-resistant manner. Viral infection promotes the interaction between ZAP and TRIM25 with these enzymes, leading to viral RNA degradation while also decreasing the abundance of many cellular transcripts. Overall, the long isoform of ZAP recruits key enzymes to assemble an RNA decay complex on viral RNA.

Candida parapsilosis is an opportunistic yeast pathogen that can cause life-threatening infections in immunocompromised humans. Whole genome sequencing (WGS) studies of the species have demonstrated remarkably low diversity, with strains typically differing by about 1.5 single nucleotide polymorphisms (SNPs) per 10 kb. However, SNP calling alone does not capture the full extent of genetic variation. Here, we define the pangenome of 372 C. parapsilosis isolates to determine variation in gene content. The pangenome consists of 5,859 genes, of which 48 are not found in the genome of the reference strain. This includes 5,791 core genes (present in ≥ 99.5% of isolates). Four genes, including the allantoin permease gene DAL4 , were present in all isolates but were truncated in some strains. The truncated DAL4 was classified as a pseudogene in the reference strain CDC317. CRISPR-Cas9 gene editing showed that removing the early stop codon (producing the full-length Dal4 protein) is associated with improved use of allantoin as a sole nitrogen source. We find that the accessory genome of C. parapsilosis consists of 68 homologous clusters. This includes 38 previously annotated genes, 27 novel paralogs of previously annotated genes and 3 uncharacterised ORFs. Approximately one-third of the accessory genome (24/68 genes) is associated with gene fusions between tandem genes in the major facilitator superfamily (MFS). Additionally, we identified two highly divergent C. parapsilosis strains and find that, despite their increased phylogenetic distance (∼30 SNPs per 10 kb), both strains have similar gene content to the other 372. Importance Candida parapsilosis is a human fungal pathogen, listed in the high priority group by the World Health Organisation. It is an increasing cause of hospital-acquired and drug-resistant infection. Here, we studied the genetic diversity of 372 C. parapsilosis isolates, the largest genomic surveillance of this species to date. We show that there is relatively little genetic variation. However, we identified two more distantly-related isolates from Germany, suggesting that even more sampling may yield more diversity. We find that the pangenome (the cumulative gene content of all isolates) is surprisingly small, compared to other fungal species. Many of the non-core genes are involved in transport. We also find that variations in gene content are associated with nitrogen metabolism, which may contribute to the virulence characteristics of this species.

Genetic functional screening technologies which identify causative genes are essential for advancing life sciences and improving drug discovery outcomes. Traditional array-based screening methods, which require significant cell numbers, face limitations when working with samples that have low proliferation capacity. While pooled library methods such as CRISPR screens can be solutions to these experimental efficiency challenges, there is still room for improvement in terms of cost and convenience. In response to these challenges, we developed PiER (Perturbation-induced intracellular events recorder) technology. PiER facilitates gene perturbation and intracellular signal detection through a novel system that integrates three DNA domains. The Perturbation domain induces gene-specific disturbances, the Response domain expresses an enzyme upon desired cellular signals, and the Memory domain records perturbation history by altering its DNA sequence via the expressed enzyme. To demonstrate PiER’s potential, we designed a vector which has a Response domain that detects WNT pathway activation. Transfecting HEK293 cells, we observed dose-dependent responses to WNT pathway activation using fluorescence microscopy and quantitative Polymerase Chain Reaction (qPCR), which confirmed successful intracellular event recording in the Memory domain. Further experiments with lentiviral PiER vectors containing a pooled shRNA library revealed the system’s capability to conduct high-throughput screening by analyzing perturbations and their effects within individual cells. PiER technology significantly enhances screening capabilities by offering a versatile and scalable approach that can be deployed without prior cell modification and single-cell isolation. Its high throughput, combined withrequiring minimal effort, presents a significant advancement for genomic research and drug target discovery.

The 7SK snRNP is a ribonucleoprotein complex comprising the non-coding RNA 7SK and the associated proteins MePCE, LARP7, and HEXIM. It regulates transcription in higher eukaryotes by sequestering the positive transcription elongation factor (P-TEFb), preventing premature entry of RNA Polymerase II in elongation. Loss of LARP7 in humans causes the Alazami syndrome, marked by restricted growth, impaired movement, and intellectual disability, though the underlying mechanisms remain unclear. In this study, we show that loss of Larp7 or 7SK RNA in Drosophila is viable but impairs locomotion and reduces axonal growth at neuromuscular junctions. Larp7 is enriched in specific motoneurons, where it functions autonomously to promote axogenesis. Reducing P-TEFb abundance partially rescues the locomotion and axonal growth defects, indicating that the 7SK complex mediates this function via transcriptional regulation. Transcriptomic analysis of mutant motoneurons revealed that the 7SK complex primarily regulates long genes with high GC content at their promoters. These findings provide new insights into the tissue-specific roles of the 7SK snRNP in transcription and organismal function.

Comparative genomic studies between contemporary and extinct hominins revealed key evolutionary modifications, but their number has hampered a system level investigation of their combined roles in scaffolding modern traits. Through multi-layered integration we selected 15 genes carrying nearly fixed sapiens -specific protein-coding mutations and developed a scalable design of combinatorial CRISPR-Cas9 bidirectional perturbations to uncover their regulatory hierarchy in cortical brain organoids. Interrogating the effects of overexpression and downregulation for all gene pairs in all possible combinations, we defined their impact on transcription and differentiation and reconstructed their regulatory architecture. We uncovered marked cell type-specific effects, including the promotion of alternative fates and the emergence of interneuron populations, alongside a core subnetwork comprising KIF15 , NOVA1 , RB1CC1 and SPAG5 acting as central regulator across cortical cell types.

The ubiquitin-proteasome system (UPS) is a fundamental regulatory mechanism maintaining cellular proteostasis through the targeted degradation of proteins. Beyond its canonical role in protein turnover, the UPS governs diverse biological processes, including cell cycle control, DNA repair, immune responses, and stress adaptation. Dysregulation of UPS components is increasingly recognized as a driving force in the pathogenesis of numerous diseases, such as cancer, neurodegenerative disorders, metabolic dysfunctions, and infections. In plants, the UPS also plays a pivotal role in environmental stress responses and hormone signaling, offering promising avenues for crop improvement. This review presents a comprehensive overview of the molecular architecture and functions of the UPS, explores its role in maintaining cellular and systemic homeostasis, and critically examines the consequences of UPS dysfunction across various disease contexts. We further highlight emerging technologies, including ubiquitinomics, CRISPR-based screens, and targeted protein degradation platforms, that are accelerating UPS research. Finally, we discuss current challenges and future opportunities for translating UPS insights into therapeutic and biotechnological innovations. A deeper understanding of the UPS across biological systems is essential for developing next-generation strategies to combat human diseases and enhance agricultural resilience.

ABSTRACT Over the past decade, Immuno-Oncology has largely focused on blocking inhibitory surface receptors like PD-1 to enhance T cell anti-tumor activity. However, intracellular immune checkpoints such as CISH, which function independently of tumor-expressed ligands, offer powerful and previously untapped therapeutic potential. As a downstream regulator of TCR signaling, CISH controls T cell activation, expansion, and neoantigen reactivity. Though historically considered undruggable, recent advances in CRISPR engineering have enabled functional interrogation of these targets. We demonstrate that CISH deletion enhances T cell activation and anti-cancer functions more effectively than other emerging intracellular checkpoints. In CAR-T cells, CISH inactivation significantly increased sensitivity to tumor antigen, enabling robust recognition and killing even at low antigen levels, conditions that often lead to treatment failure with conventional T cell therapies, mirroring antigen escape scenarios seen in solid tumors. Our findings further validate CISH as a potent and druggable intracellular checkpoint capable of boosting anti-tumor T cell responses across diverse cancer types, independent of PD-L1 status. The underlying mechanisms of CISH inhibition may help explain the positive outcomes reported in recent clinical studies of this approach in solid tumor immunotherapy.

Glandular trichomes are specialized epidermal structures that play an essential role in plant defense by synthesizing, storing, and secreting specialized metabolites. This study investigates the function of NtAGL66 , an AGAMOUS-like gene in Nicotiana tabacum , uncovering its role in the development of secretory heads in long glandular trichomes. Expression profiling reveals that NtAGL66 is specifically expressed in the developing secretory glands. Functional analyses show that NtAGL66 overexpression promotes the differentiation of the secretory structure, while CRISPR-Cas9-mediated knockout significantly reduces the capacity of trichomes to form functional secretory glands, highlighting its essential role in trichome specialization. Transcriptomic (RNA-seq) and functional genomic (DAP-seq) analyses indicate that NtAGL66 regulates also secondary metabolic pathways and is likely involved in broader transcriptional networks, including floral development. Notably, this includes genes such as NtTOE1, previously shown to control both floral organogenesis and glandular trichome formation in tomato. Moreover, NtAGL66 directly regulates the transcription factor NtGL2 through promoter binding. By identifying an AGAMOUS-like gene as a key regulator of secretory gland development, this study offers novel insights into the genetic mechanisms underlying glandular trichome differentiation and specialized metabolite biosynthesis in Solanaceae .

Summary CRISPR-Cas systems provide adaptive immunity against phage infection in prokaryotes using an RNA-guided complex that recognizes complementary foreign nucleic acids. Different types of CRISPR-Cas systems have been identified that differ in their mechanism of defense. Upon infection, Type III CRISPR-Cas systems employ the Cas10 complex to find phage transcripts and synthesize cyclic oligo-adenylate (cOA) messengers. These ligands bind and activate CARF immune effectors that cause cell toxicity to prevent the completion of the viral lytic cycle. Here we investigated two proteins containing an N-terminal haloacid dehalogenase (HAD) phosphatase domain followed by four predicted transmembrane helices and a C-terminal CARF domain, which we named Chp. We show that, in vivo, Chp localizes to the bacterial membrane and that its activation induces a growth arrest, leads to a depletion of ATP and IMP and prevents phage propagation during the type III CRISPR-Cas response. In vitro, the CARF domain of Chp binds cyclic tetra-adenylates and the HAD phosphatase domain dephosphorylates dATP, ATP and IMP. Our findings extend the range of molecular mechanisms employed by CARF effectors to defend prokaryotes against phage infection.

The natural context in which CRISPR-Cas systems are active in Enterobacteriaceae has remained enigmatic. Here, we find that the Citrobacter rodentium Type I-E CRISPR-Cas system is activated by the oxygen-responsive transcriptional regulator Fnr in the anoxic mouse intestine. Since Fnr-dependent regulation is predicted in ~41% of Enterobacteriaceae cas3 orthologs, we propose that anoxic regulation of CRISPR-Cas immunity is an adaptation that protects Enterobacteriaceae against threats arising from the intestinal microbiome.

Many taxa have independently evolved genetic sex determination where a single gene located on a sex chromosome controls gonadal differentiation. The gene anti-Mullerian hormone (amh) has convergently evolved as a sex determination gene in numerous vertebrate species, but how this gene has repeatedly evolved this novel function is not well understood. In the threespine stickleback (Gasterosteus aculeatus), amh was duplicated onto the Y chromosome (amhy) ~22 million years ago. To determine whether amhy is the primary sex determination gene, we used CRISPR/Cas9 and transgenesis to show that amhy is necessary and sufficient for male sex determination, consistent with the function of a primary sex determination gene. While we find substantial regulatory evolution has occurred in amhy, we did not observe an increase in amhy expression across early development relative to its autosomal paralog, amha or a significant difference in total amh dosage between males and females around the time of sex determination. This indicates the mechanism of sex determination may involve subtle changes in cell-specific expression or coding sequence evolution. The creation of sex reversed lines also allowed us to investigate the genetic basis of secondary sex characteristics. Threespine stickleback have striking differences in behavior and morphology between sexes. Here we show one of the classic traits important for reproductive success, blue male nuptial coloration, is controlled by both Y-linked genetic factors as well as hormonal factors independent of sex chromosome genotype. This research establishes stickleback as a model to investigate how amh regulates gonadal development and how this gene repeatedly evolves novel function in sex determination. Analogous to the 'four core genotypes' model in house mice, sex-reversed threespine stickleback offer a new vertebrate model for investigating the separate contributions of gonadal sex and sex chromosomes to sexual dimorphism.

The typhoid toxin is a secreted virulence factor of typhoidal serovars of the bacterial pathogen Salmonella enterica implicated in typhoid fever and chronic infections. The toxin causes a DNA damage response in human cells, characterised by cell-cycle arrest and cellular distension, resulting in cellular senescence and increased bacterial burden. To better understand host responses to typhoid toxin, we performed a transcriptomic analysis of intoxicated host cells and found that the toxin induced expression of genes relating to the type-I interferon response, including the ubiquitin-like protein ISG15. ISG15 was upregulated in a STING-dependent manner, reduced bacterial burden, and was found to be critical to host cell survival in response to the typhoid toxin and purified interferon. This highlights ISG15 as an important component of the host cell defence to the typhoid toxin.

Genome alterations arise from inaccurate DNA repair, accumulating into distinct mutational signatures. Here, we investigate the role of every genomically encoded gene in double-strand break (DSB) repair by generating high-resolution outcome profiles following gene knockouts. Using a CRISPR/Cas9-based, massive-parallel bulk library approach, we construct a comprehensive, user-explorable mutational signature catalogue (MUSIC), mapping the full repertoire of DSB repair factors. Our analysis identifies and validates gene clusters – including nearly all known and several novel genes – linked to non-homologous end-joining, 53BP1 sub-pathways, homology-directed repair, and polymerase Theta (POLQ)-mediated end-joining. By focusing on pathway-specific repair outcomes, we uncover a previously unrecognized role for the WRN helicase in suppressing inverted templated insertions, a poorly understood POLQ-associated mutational signature also found in human disease alleles. Furthermore, in-depth analysis of MUSIC’s scar features reveals unexpected distinctions among genes within the same pathway, providing mechanistic insight and opening multiple new avenues for investigation into chromosomal break repair.

Abstract Before human genome sequencing, a genome-wide study of sibling centenarian pairs identified a longevity-associated locus on chromosome 4. Here, we mapped the genes in this locus and identified a collagen gene, COL25A1. Introducing an SNP linked to longevity that changes a serine predicted to be phosphorylated to leucine in COL25A1 , into col-99 , the C. elegans ortholog, extended lifespan. These col-99(gk694263 [S106L] ) SNP-mutants exhibited enhanced innate immune-related transcriptional responses, and their lifespan extension was abolished by inhibiting the p38 MAPK pathway. YAP-1, a transcriptional co-activator responsive to extracellular matrix changes, was essential for this longevity. Mechanistically, we propose that this SNP modifies furin-mediated cleavage of this transmembrane collagen in vitro, and expressing the cleaved extracellular domain of COL-99 alone was sufficient to prolong lifespan. These findings reveal a potential mechanism by which a human centenarian-associated SNP in COL25A1 influences furin cleavage and shedding of the collagen ectodomain to promote healthy longevity.

Basement membranes (BMs) are specialized extracellular matrices (ECMs) essential for tissue structure and function. In non-vertebrates, ECM components can be produced both locally and by distant tissues. In contrast, mammalian ECM has traditionally been considered to originate predominantly from adjacent or tissue-resident cells. The kidney glomerular basement membrane (GBM), composed of laminin-α5β2γ1 and collagen-α3α4α5(IV), is produced by neighboring epithelial cells and functions as a filtration barrier. Alport syndrome, a genetic kidney disease in children, is characterized by GBM structural defects and ectopic laminin-α2 deposition, but the source of this laminin remains unknown. Here, using CRISPR/Cas9 transgenic models, we demonstrated that ectopic laminin-α2 originates not from local kidney cells but from the circulation. Furthermore, laminin-α2 in the mesangium partially derives from circulating sources even under healthy conditions. Our findings uncover a non-cell-autonomous mechanism whereby GBM integrity regulates circulating protein incorporation, revealing a previously unrecognized trans-tissue regulation of BM composition in mammals.

When cells divide, the newly replicated sister chromatids must be segregated evenly to the daughter cells. During mitosis, mechanical force is applied by spindle microtubules in 2 ways: first by pushing on chromosome arms to promote chromosome congression to the cell equator in metaphase, and then by pulling on kinetochores to promote sister chromatid disjunction during anaphase. For segregation to proceed faithfully, the pliable interphase chromatin must be transformed into stiff mitotic chromosomes able to withstand these forces. However, it is unclear how the cell establishes chromosome stiffness and what the consequences are for dividing cells if this stiffness is disrupted. Many of the structural changes imposed on chromosomes in mitosis are driven by Condensin complexes, in conjunction with Topoisomerase IIα. Here, we have combined rapid protein depletion and live cell imaging with in-depth mechanical characterization of purified mitotic chromosomes to probe the roles of Condensins I and II in the establishment and maintenance of the mechanical strength of mitotic chromosomes. We show that Condensin I, but not Condensin II, is required to establish chromosome stiffness and chromatin elasticity, and yet ceases to be required for the maintenance of these properties once chromosome formation has been completed. Nevertheless, depletion of Condensin I from already formed chromosomes still impacts centromeric chromatin and leads to a loss of sister centromere cohesion. We propose that the extensive chromatin loop network established by Condensin I is locked in place by Topoisomerase IIα mediated DNA catenation.

The biopharmaceutical sector relies on CHO cells to investigate biological processes and as the preferred host for production of biotherapeutics. Simultaneously, advancements in CHO cell genome assembly have provided insights for developing sophisticated genetic engineering strategies. While the majority of these efforts have focused on coding genes, with some interest in transcribed non-coding RNAs (e.g., microRNAs and lncRNAs), there remains a lack of genome-wide systematic studies that precisely examine the remaining 90% of the genome. This unannotated “dark matter” includes regulatory elements and other, poorly understood or characterized functionality of the genome that may be potentially critical for cell survival. In this study, we deployed a genome-scale CRISPR screening platform with 112,272 paired guide RNAs targeting 14,034 genomic regions for complete deletion of 150 kb long sections. This platform enabled the execution of a negative screen that selectively identified dying cells to determine regions essential for cell survival. By using paired gRNAs, we overcame the intrinsic limitations of traditional frameshift strategies, which will likely have little or no effect on the non-coding genome. This study revealed 427 regions essential for CHO survival, many of which currently lack gene annotation or known function. For these regions we present annotation status, transcriptional activity as well as annotated chromatin states such as enhancers. Selected regions, specifically those that were negative for all the above, were individually deleted for confirmation. This work sheds a novel light on a substantial part of the mammalian genome which is traditionally difficult to investigate and therefore, neglected.

SUMMARY Metabolites are essential substrates for epigenetic modifications. Although nuclear acetyl-CoA constitutes a small fraction of the whole cell pool, it regulates cell fate by locally providing histone acetylation substrate. Here, we combined phenotypic chemical screen and genome-wide CRISPR screen to demonstrate a nucleus-specific acetyl-CoA regulatory mechanism that can be modulated to achieve therapeutic cancer cell reprogramming. While previously thought that nucleus-localized pyruvate dehydrogenase complex (nPDC) is constitutively active, we found that nPDC is constitutively inhibited by the nuclear protein ELMSAN1 through direct interaction. Pharmacologic inhibition of the ELMSAN1-nPDC interaction derepressed nPDC activity, enhancing nuclear acetyl-CoA generation and reprogramming cancer cells to a postmitotic state with diminished cell-of-origin signatures. Reprogramming was synergistically enhanced by histone deacetylase 1/2 inhibition, resulting in inhibited tumor growth, durably suppressed tumor-initiating ability, and improved survival in multiple cancer types in vivo , including therapy-resistant sarcoma patient-derived xenografts and carcinoma cell line xenografts. Our findings highlight the potential of targeting ELMSAN1-nPDC as epigenetic cancer therapy.

Despite the availability of numerous methods for controlling gene expression, there remains a strong need for technologies that maximize two key properties: selectivity and reversibility. To this end, we have developed a novel approach that exploits the highly sequence-specific nature of CRISPR-associated endoribonucleases (Cas RNases), which recognize and cleave short RNA sequences known as direct repeats (DRs). In this approach, referred to as DREDGE (direct repeat-enabled downregulation of gene expression), selective control of gene expression is enabled by introducing one or more DRs into the untranslated regions (UTRs) of target mRNAs, which will then be cleaved upon expression of the cognate Cas RNase. We previously demonstrated that the expression of target genes with DRs in their 3' UTRs are efficiently controlled by the DNase-dead version of Cas12a (dCas12a) with a high degree of selectivity and complete reversibility. Here we assess the feasibility of using DREDGE to regulate the expression of genes with DRs inserted within their 5' UTRs. Among five different Cas RNases tested, Csy4 was found to be the most efficient in this format, yielding robust downregulation with rapid onset in doxycycline-regulatable systems targeting either a stably expressed fluorescent protein or an endogenous gene, notably in a fully reversible manner. Unexpectedly, dCas12a was also found to be modestly effective despite binding essentially irreversibly to the cut mRNA on its 5' end and boosting mRNA levels. Our results expand the utility of DREDGE as an attractive method for regulating gene expression in a targeted, highly selective, and fully reversible manner.

ABSTRACT Type III CRISPR systems detect non-self RNA and activate the enzymatic Cas10 subunit, which generates nucleotide second messengers for activation of ancillary effectors. Although most signal via cyclic oligoadenylate (cOA), an alternative class of signalling molecule SAM-AMP, formed by conjugating ATP and S-adenosyl methionine, was described recently. SAM-AMP activates a trans-membrane effector of the CorA magnesium transporter family to provide anti-phage defence. Intriguingly, immunity also requires SAM-AMP degradation by means of a specialised CRISPR-encoded NrN family phosphodiesterase in Bacteroides fragilis . In Clostridium botulinum , the nrn gene is replaced by a gene encoding a SAM-AMP lyase. Here, we investigate the structure and activity of C. botulinum SAM-AMP lyase, which can substitute for the nrn gene to provide CorA-mediated immunity in Escherichia coli . The structure of SAM-AMP lyase bound to its reaction product methylthioadenosine-AMP (MTA-AMP) reveals key details of substrate binding and turnover by this PII superfamily protein. Bioinformatic analyses reveal candidate phage-encoded SAM-AMP lyases and we demonstrate that one, hereafter named AcrIIIB4, degrades SAM-AMP efficiently in vitro .

Summary Transcriptional complexes with a common composition regulate the production of flavonoid pigments, trichomes, root hairs and other epidermal traits in seed plants. These complexes are composed of transcription factors from the MYB and basic helix-loop-helix (bHLH) families along with a tryptophan-aspartate repeat (WDR) scaffold protein (MBW complexes). The MYB member has been found to be the most pathway-specific component of the complex and modifications to these MYB genes are overrepresented in studies investigating the genetic basis of changes in pigmentation phenotypes across flowering plants. Here we investigated the orthologues of the MBW complex in a divergent lineage to understand its origin and evolution. We found evidence that these transcriptional complexes also form in the liverwort Marchantia polymorpha , indicating, together with an analysis of published gene family phylogenies, that they are ancestral to land plants. The functions of each of the two orthologous MYB genes, Mp MYB14 and Mp MYB02 , both depend on the single orthologous bHLH gene, Mp bHLH12 . We could not assess the functional role of the WDR genes in M. polymorpha , due to low mutant recovery suspected to be caused by pleiotropic effects on viability. We propose that the two transcriptional complexes with alternative MYB paralogues in M. polymorpha represent an ancestral function, regulation of the flavonoid pathway, and a derived function, maturation of liverwort-specific oil bodies. Our findings imply a replicated pattern by which new complexes have evolved in independent land plant lineages, through duplication of the evolutionarily labile MYB member and co-option of its interaction partners.

Abstract Background: The CD247 chain of the T-cell receptor (TCR) is essential for normal T cell development and function. Reported CD247-deficient patients showed severe immunodeficiency despite the presence of two populations of peripheral T cells, most with low TCR levels carrying the germline variant and a few with higher TCR levels due to somatic reversion. However, the revertant T cells remained a minority and did not improve the patients’ clinical status. Purpose: To compare the capability of somatic reversions of CD247 germline changes (p.M1T and p.Q70X) to restore TCR expression and function. Methods: CD247 wild-type (WT) and p.Q70L/W/Y somatic revertants were individually introduced in CD247-deficient mouse (MA5.8), human mutant (PM1T), and CRISPR/Cas9-generated Jurkat (ZKO) T cell lines by nucleofection or transduction. Results: MA5.8 mouse T cells do not accurately model human CD247 deficiencies, as Q70X restores TCR expression in MA5.8 but not in human cells. In human cell models, all somatic revertant variants restored TCR expression with varying degrees (WT=Q70L>Q70W>Q70Y). However, rescue of TCR-induced activation events, including ZAP-70 phosphorylation and CD69/CD25 upregulation, did not match such hierarchy (WT=Q70W>Q70L=Q70Y). Conclusion: Somatic reversions, such as those detected in patients with pathogenic CD247 germinal changes, display a discordant capability to rescue TCR expression versus function. These findings shed light on the role of CD247 in TCR expression and function during human T cell development, with implications for immunodeficiencies, as well as for the biological consequences of CD247 somatic mosaicism.

Abstract Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), driven by virulence factors such as iron acquisition systems and adhesive pili. In this study, we employed CRISPR-Cas9-mediated genome editing to functionally inactivate two critical virulence genes— iucD , involved in aerobactin-mediated iron uptake, and papC , encoding the outer membrane usher protein essential for P pilus assembly. Using a clinical UPEC isolate, we introduced premature stop codons via homologous repair templates guided by gene-specific single-guide RNAs. Colony PCR and Sanger sequencing confirmed precise site-specific editing, leading to truncated protein variants. In silico analyses using InterPro and Swiss-Model revealed a complete loss of essential domains in both proteins. Molecular docking studies demonstrated a marked reduction in binding affinities of truncated IucD for NAD(P)H and impaired protein-protein interaction between truncated PapC and PapG. This study highlights the utility of CRISPR-Cas9 as a powerful tool for dissecting bacterial pathogenesis and supports the potential of targeting virulence determinants like iucD and papC as part of an antivirulence strategy for managing UPEC infections.

Abstract The efficacy of chimeric antig en receptor (CAR) T cell therapy in solid tumors is limited by immunosuppression and antigen heterogeneity. To overcome these barriers, “armored” CAR T cells, which secrete proinflammatory cytokines, have been developed. However, their clinical application has been limited due to toxicities related to peripheral expression of the armoring transgene. Here, we developed a novel CRISPR knock-in strategy that uses endogenous gene regulatory mechanisms to drive transgene expression in a tumor-localized manner. By screening endogenous genes with tumor-restricted expression, the NR4A2 and RGS16 promoters were identified to support the delivery of cytokines such as IL-12 and IL-2 directly to the tumor site, leading to enhanced anti-tumor efficacy and long-term survival of mice in both syngeneic and xenogeneic models. This was concomitant with improved CAR T cell polyfunctionality, activation of endogenous anti-tumor immunity, a favorable safety profile, and was applicable using CAR T cells from patients.