Current standard food detection methods do not distinguish between infectious and non-infectious norovirus leading to uncertainty in the interpretation and management of a norovirus positive food sample. These methods also require expensive RT-qPCR based equipment. In contrast, CRISPR-based, compared to RT-qPCR based, detection methods yield similar sensitivity and specificity and are generally less expensive. The aim of this study was to detect norovirus with an intact capsid, a proxy for infectivity, through a CRISPR-Cas13a based detection method in conjunction with acapsid integrity assay. Our CRISPR method detected murine norovirus (MNV-1), with an intact capsid, at a limit of detection of 2.59 log10 gc/ 25 g (5 gc/ rx). This method did not cross-react with other targets (synthetic hepatitis A virus; human norovirus GI, GII; rotavirus). Compared with RT-qPCR, this CRISPR based method showed an increased sensitivity when detecting low copy numbers of RNase-pretreated MNV-1 in lettuce and blueberries samples. This is the first report describing a CRISPR-based detection of potentially infectious viruses in food samples.

The PI3K/AKT signaling pathway is frequently dysregulated in cancer and controls key cellular processes such as survival, proliferation, metabolism and growth. Protein glycosylation is essential for proper protein folding and is also often deregulated in cancer. Cancer cells depend on increased protein folding to sustain oncogene-driven proliferation rates. The N-glycosyltransferase asparagine-linked glycosylation 3 homolog (ALG3), a rate-limiting enzyme during glycan biosynthesis, catalyzes the addition of the first mannose to glycans in an alpha-1,3 linkage. Here we show that ALG3 is phosphorylated downstream of the PI3K/AKT pathway in both growth factor-stimulated cells and PI3K/AKT hyperactive cancer cells. AKT directly phosphorylates ALG3 in the amino terminal region at Ser11/Ser13. CRISPR/Cas9-mediated depletion of ALG3 leads to improper glycan formation and induction of endoplasmic reticulum stress, the unfolded protein response, and impaired cell proliferation. Phosphorylation of ALG3 at Ser11/Ser13 is required for glycosylation of cell surface receptors EGFR, HER3 and E-cadherin. These findings provide a direct link between PI3K/AKT signaling and protein glycosylation in cancer cells.

The leading region of conjugative plasmids is the first to enter recipient cells during conjugation. This region is enriched in anti-defence genes, suggesting that defence system evasion shapes plasmid organization. Might this selective pressure also variably impact sequence composition? Here we hypothesize that the leading region is subject to stronger selection to avoid triggering defence systems. We investigate this for Type II restriction-modification (RM) systems, using a dataset of thousands of conjugative plasmids belonging to different plasmid taxonomic units (PTUs). Consistent with our hypothesis, we find evidence of RM target depletion in leading regions, despite higher average GC-content in the leading region which should increase palindrome density. We find evidence of strand-specific nucleotide skews that could cause this depletion, which we suggest could have drivers beyond RM evasion. Our work opens up intriguing questions about how the sequence composition of the leading region has been shaped by different selective pressures.

First identified in 2009, Candidozyma auris (formerly Candida auris ) is an emerging multidrug resistant fungus that can cause invasive infections with a crude mortality rate ranging from 30-60%. Currently, 30-50% of C. auris isolates are intrinsically resistant to amphotericin B. In this work, we characterized a clinical case of acquired amphotericin B resistance using whole genome sequencing, a large-scale phenotypic screen, comprehensive sterol profiling, and genotypic reversion using CRISPR. Data obtained in this work provides evidence that a deletion resulting in a frameshift in ERG3 contributes to the observed resistant phenotype. Characterization of this isolate also revealed a fitness cost is associated with the abrogation of ergosterol production and its replacement with other late-stage sterols. This article presents a clinical case description of amphotericin B resistance from a frameshift mutation in ERG3 in C. auris and marks an advancement in the understanding of antifungal resistance in this fungal pathogen.

Gene discovery studies in individuals with diabetes diagnosed within 6 months of life (neonatal diabetes, NDM) can provide unique insights into the development and function of human pancreatic beta-cells. We describe the identification of homozygous PAX4 loss-of-function variants in 2 unrelated individuals with NDM: a p.(Arg126*) stop-gain variant and a c.-352_104del deletion affecting the first 4 PAX4 exons. We confirmed the p.(Arg126*) variant causes nonsense mediated decay in CRISPR-edited human induced pluripotent stem cell (iPSC)-derived pancreatic endoderm cells. Integrated analysis of CUT&RUN and RNA-sequencing in PAX4 -depleted islet cell models identified genes directly regulated by PAX4 involved in both pancreatic islet development and glucose-stimulated insulin secretion. Both probands had transient NDM which remitted in early infancy but relapsed between the ages of 2 and 7 years, demonstrating that in contrast to mouse models, PAX4 is not essential for the development of human pancreatic beta-cells.

ABSTRACT CAR-T cell therapies are revolutionizing the treatment of refractory and relapsed haematological malignancies, but many patients do not exhibit long-term responses, and these therapies are less effective against solid tumors. Poor persistence of CAR-T cells in patients is associated with therapeutic failure, highlighting the need to identify strategies promoting in vivo expansion. Here, we developed an in vivo competitive screening method to identify genes whose inactivation confers a selective advantage to CAR-T cells. Inactivation of 50 genes in a heterogeneous population of T cells expressing an EGFR-targeting CAR revealed that disruption of REGNASE-1, SOCS1, PTPN2 , and P16/NK4A conferred a selective advantage to CAR-T cells in human lung tumor-bearing mice. Consistently, inactivation of these genes improved tumor eradication by CAR-T cells. Interestingly disruption of other genes, described to improve CAR-T cell function in other contexts, had a negative impact in this orthotopic lung tumor model. Further evaluation of long-term effects in a subcutaneous model, highlighted SOCS1 ablation as the most promising strategy for in vivo CAR-T cell amelioration. These results support the importance of evaluating CAR-T cell editing strategies in tumor-specific models and highlight the versatility of our screening approach as a pre-clinical tool for context-specific studies on CAR-T cells amelioration.

Cell migration plays a key role in normal developmental programs and in disease, including immune responses, tissue repair, and metastasis. Unlike other cell functions, such as proliferation which can be studied using high-throughput assays, cell migration requires more sophisticated instruments and analysis, which decreases throughput and has led to more limited mechanistic advances in our understanding of cell migration. Current assays either preclude single-cell level analysis, require tedious manual tracking, or use fluorescently labeled cells, which greatly limit the number of extracellular conditions and molecular manipulations that can be studied in a reasonable amount of time. Using the migration of cancer cells as a testbed, we established a workflow that images large numbers of cells in real time, using a 96-well plate format. We developed and validated a machine-vision and deep-learning analysis method, DeepBIT, to automatically detect and track the migration of individual cells from time-lapsed videos without cell labeling and user bias. We demonstrate that our assay can examine cancer cell motility behavior in many conditions, using different small-molecule inhibitors of known and potential regulators of migration, different extracellular conditions such as different contents in extracellular matrix and growth factors, and different CRISPR-mediated knockouts. About 1500 cells per well were tracked in 840 different conditions, for a total of ~1.3M tracked cells, in 70h (5 min per condition). Manual tracking of these cells by a trained user would take ~5.5 years. This demonstration reveals previously unidentified molecular regulators of cancer cell migration and suggests that collagen content can change the sign of how cytoskeletal molecules can regulate cell migration.

Stem cell populations in tissues require precise regulation of their number and quality to maintain proper organ growth and regenerative capacity. Amongst various regulatory mechanisms, immune cells are emerging to directly regulate stem cell populations. The medaka retinal stem cell niche, a model for lifelong neurogenic growth, provides a system to study immune cell-stem cell interactions. Here we investigate how microglia, the resident macrophages of the central nervous system, regulate the retinal stem cell niche. We identify that bona fide retinal stem cells express the chemokine ccl25b while its cognate receptor, ccr9a, is expressed in microglia. These microglia form a static surveillance ring adjacent to the stem cell niche and actively phagocytose retinal stem cells. We show that targeted mutation of ccl25b affects microglia mobility and leads to reduction of retinal-stem cell-derived material in microglia. Interference with microglia by deletion of spi1b reveals that microglia absence leads to increased numbers of ccl25b-positive stem cells and results in morphological defects in the retinal stem cell niche and retina. Overall, our data show that under homeostatic conditions the retinal stem cell population, essential for proper eye development, is actively pruned by immune surveillance.

Centromeres in eukaryotes are defined by the presence of histone H3 variant CENP-A/CENH3. Chlamydomonas encodes two predicted CENH3 paralogs, CENH3.1 and CENH3.2, that have not been previously characterized. We generated peptide antibodies to unique N-terminal epitopes for each of the two predicted Chlamydomonas CENH3 paralogs as well as an antibody against a shared CENH3 epitope. All three CENH3 antibodies recognized proteins of the expected size on immunoblots and had punctuated nuclear immunofluorescence staining patterns. These results are consistent with both paralogs being expressed and localized to centromeres. CRISPR-Cas9 mediated insertional mutagenesis was used to generate predicted null mutations in either CENH3.1 or CENH3.2 . Single mutants were viable but cenh3.1 cenh3.2 double mutants were not recovered, confirming that the function of CENH3 is essential. We sequenced and assembled two chromosome-scale Chlamydomonas genomes from strains CC-400 and UL-1690 (a derivative of CC-1690) with complete centromere sequences for 17/17 and 14/17 chromosomes respectively, enabling us to compare centromere evolution across four isolates with near complete assemblies. These data revealed significant changes across isolates between homologous centromeres including mobility and degeneration of ZeppL-LINE1 (ZeppL) transposons that comprise the major centromere repeat sequence in Chlamydomonas. We used Cleavage Under Targets and Tagmentation (CUT&Tag) to purify and map CENH3-bound genomic sequences and found enrichment of CENH3-binding almost exclusively at predicted centromere regions. An interesting exception was chromosome 2 in UL-1690, which had enrichment at its genetically mapped centromere repeat region as well as a second, distal location, centered around a single recently acquired ZeppL insertion. The CENH3-bound regions of the 17 Chlamydomonas centromeres ranged from 63.5kb (average lower estimate) to 175kb (average upper estimate). The relatively small size of its centromeres suggest that Chlamydomonas may be a useful organism for testing and deploying artificial chromosome technologies.

CEP290 is an important human disease gene, as mutations are implicated in a broad spectrum of autosomal recessive ciliopathies, including Leber congenital amaurosis and Joubert, Meckel, Senior-LØken or Bardet Biedl syndromes. To create isogenic mutant human induced pluripotent stem cell (hiPSC) lines for disease modeling, we employed CRISPR/Cas9 to introduce disease-relevant mutations into the control hiPSC line HMGU1 (ISFi001-A). Thorough characterization of the lines, including the effect of the mutation at the mRNA and protein level, shows that these CEP290 -mutant lines provide a useful resource for studying ciliopathy disease mechanisms and cilia biology through differentiation into diverse cell types and organoids.

CRISPR–Cas systems are adaptive immune mechanisms in bacteria and archaea that protect against invading genetic elements by integrating short fragments of foreign DNA into CRISPR arrays. These arrays consist of repetitive sequences interspersed with unique spacers, guiding Cas proteins to recognize and degrade matching nucleic acids. The integrity of these repeat sequences is crucial for the proper function of CRISPR–Cas systems, yet their mutational dynamics remain poorly understood. In this study, we analyzed 56,343 CRISPR arrays across 25,628 diverse prokaryotic genomes to assess the mutation patterns in CRISPR array repeat sequences within and across different CRISPR subtypes. Our findings reveal, as expected to some extent, that mutation frequency is substantially higher in terminal repeat sequences compared to internal repeats consistently across system types. However, the mutation patterns exhibit an unexpected amount of variation among different CRISPR subtypes, suggesting that selective pressures and functional constraints shape repeat sequence evolution in distinct ways. Understanding these mutation dynamics provides insights into the stability and adaptability of CRISPR arrays across diverse bacterial and archaeal lineages. Additionally, we elucidate a novel relationship between repeat mutations and spacer dynamics, demonstrating that hotspots for terminal repeat mutations coincide with regions exhibiting spacer conservation. This observation corroborates recent findings by Fehrenbach et al. (2024) indicating that spacer deletions occur at a frequency 374 times greater than that of mutations and are significantly influenced by repeat misalignment. Our findings suggest that repeat mutations play a pivotal role in spacer retention or loss, or vice versa, thereby highlighting an evolutionary trade-off between the stability and adaptability of CRISPR arrays.

Colorectal cancer (CRC) remains a major global health concern, partly due to resistance to therapy and the lack of new effective treatments for advanced disease. The combination of 5-Fluorouracil (5FU, a thymidylate synthase inhibitor) and irinotecan (a topoisomerase 1 inhibitor) is widely used in first-line and subsequent treatments. This study aimed to identify novel therapeutic targets to enhance combinatorial therapy, improving treatment efficacy and durability of response. We performed a loss-of-function screen using HT29 CRC cell line and a retroviral library containing 7296 shRNAs targeting 912 chromatin genes. Cells were then treated with 5FU and SN38 (the active metabolite of irinotecan) or left untreated for 4 weeks. Genes enriched in resistant clones were identified through next-generation sequencing. Among candidate genes, PARG was selected for functional validation. CRISPR/Cas9-mediated knockout (HT29 PARG-KO) resulted in increased global poly(ADP-ribosyl)ation after 5FU and SN38 treatment. PARG depletion led to reduced cell viability and increased apoptosis, particularly after 5FU exposure. Pharmacological PARG inhibition (PDD00017273) synergized with 5FU and SN38 across three CRC models (HT29, DLD1, HT115). In vivo , HT29 PARG-KO xenografts were more sensitive to 5FU. Immunohistochemical analysis of 170 CRC patient tumors revealed that positive PARG expression correlated with poor response to 5FU + Irinotecan, increased liver metastases, and worse long-term survival. Our findings highlight PARG as a promising therapeutic target for CRC, where its inhibition enhances the efficacy of standard chemotherapy.

Epigenetic modifications play a crucial role in gene regulation and have been implicated in various physiological processes and disease conditions. DNA methylation (DNAm) has been implicated in the etiology and progression of many stress-related psychiatric behaviors such as depression. The ability to manipulate DNAm may provide a means to reverse and treat such disorders. Although CRISPR-based technologies have enabled locus-specific DNAm editing, their clinical applicability may be limited due to immunogenicity concerns and off-target effects. In this study, we introduce a novel approach for targeted DNAm manipulation using single-stranded, methylated DNA probes. We designed probes targeting the glucocorticoid response element (GRE) within the FKBP5 (FK506 binding protein 5) gene, a key regulator of stress response and depressive symptoms, and the promoter region of the MAOA (monoamine oxidase A) gene. In both HEK293 human embryonic kidney and mouse pituitary AtT-20 cells, transfection with their respective methylated probes significantly increased DNAm at targeted CpG sites in a persistent and dose-dependent manner. Importantly, the induced methylation effectively attenuated glucocorticoid-induced upregulation of FKBP5 gene expression. Alteration of methylation was specific to single-stranded probes, as double-stranded methylated probes and unmethylated probes showed no significant effects. Our findings suggest that methylated DNA probes have the potential to function as a simple tool for targeted epigenetic manipulation and serve as a safer alternative to CRISPR-based epigenome editing tools for the treatment of stress-related disorders such as depression.

Abstract Background Propionic acidemia (PA) is a rare autosomal recessive metabolic disorder caused by defects in propionyl-CoA carboxylase (PCC), a mitochondrial enzyme composed of six alpha (PCCA) and six beta (PCCB) subunits. Mutations in PCCA/PCCB genes disrupt PCC function, leading to toxic metabolite accumulation and clinical manifestations. Current research is limited by inadequate patient-derived cellular models and ethical constraints in sample acquisition. Method Using CRISPR/Cas9-mediated gene editing, we established an isogenic human induced pluripotent stem cell (iPSC) line carrying the PCCA c.2002G > A mutation. Results The mutant iPSCs showed sustained expression of pluripotency markers (OCT4, NANOG, SOX-2), maintained normal karyotype (46, XY), and retained trilineage differentiation capacity. Functional characterization demonstrated significantly reduced PCC enzyme activity, accurately modeling PA metabolic pathology. Conclusions This isogenic iPSC line provides an ethically unconstrained platform to investigate PA molecular mechanisms and genotype-phenotype relationships. The model enables systematic drug screening and therapeutic development while overcoming patient sample limitations.

The CRISPR/Cas9 system, derived from the adaptive immune defence of bacteria and archaea, has emerged as a powerful tool for genome engineering by enabling precise, site-specific modifications. Type II bacterial CRISPR systems recognize and cleave DNA in various microorganisms using the RNA-guided Cas9 endonuclease. We present a streamlined and efficient approach for genome editing in two strains of Saccharomyces cerevisiae , focused on targeted mutagenesis of the ADE 2 gene of the BY4741 strain and homology-directed repair of the ade2 locus of the W303-1A strain, involving a single-step transformation strategy utilizing the pML104 plasmid, which encodes the Cas9 endonuclease and a user-defined single-guide RNA (gRNA) complementary to the target site. In the BY4741 (wildtype) strain, pML104-gRNA components achieved successful ADE2 disruption rates of 40% (95% CI: 0.10–0.70), confirmed by adenine auxotrophy screening and Sanger sequencing. Additionally, co-transformation of gRNA plasmid and donor repair templates in the W303-1A ( ade2 mutant) strain also resulted in 40% recombination efficiency, restoring the function of the mutant gene to allow activation of de novo purine biosynthesis activity when cultured on SC-Ade medium. This optimized, cost-effective CRISPR/Cas9 method enhances editing efficiency in S. cerevisiae , providing a robust and practical platform for high-precision genetic modifications with broad applications in molecular biology and biotechnology.

Protein modification by interferon-stimulated gene 15 (ISG15), termed ISGylation, exhibits antiviral properties and influences tumorigenesis, genome stability and metabolic processes. ISGylation is counteracted by the specific protease USP18. Likewise, viral proteases such as the papain-like protease (PLpro) from SARS-CoV-2 cleave ISG15 to undermine the host immune response. Beyond its role as a deISGylating enzyme, USP18 acts as a major negative regulator of the IFN signaling pathway in a STAT2-dependent manner. In humans, unconjugated ISG15 secures USP18 stability and the absence of USP18 or impaired STAT2/USP18 binding cause fatal interferonopathies. Thus, the USP18 signaling hub represents a critical checkpoint for type I IFN signaling and ISGylation, qualifying it as a promising immune and cancer drug target. However, suitable assays to monitor protein-protein interactions (PPIs) within the USP18/ISG15/STAT2 signaling hub and to screen for PPI modulators are missing and no specific inhibitors targeting USP18 interactions are available. To address this gap, we developed a method based on the NanoLuc luciferase (NLuc) Bioluminescence Energy Transfer (NanoBRET) assay system to study PPIs. Firstly, we generated stable cell lines suitable to monitor USP18/ISG15 and USP18/STAT2 interactions, providing a semi high-throughput screening (HTS)-compatible platform. In combination with a virtual pre-screen of 60,000 compounds against USP18 in silico , this assay allowed us to identify a first small molecule (ZHAWOC8655) that compromises cellular USP18/ISG15 binding and inhibits USP18 protease activity in vitro . To further explore the potential of using the NanoBRET system for testing PPI modulators, we evaluated the effect of GRL0617, a compound which was shown to disrupt the interaction between SARS-CoV-2 PLpro/ISG15 as well as SARS-CoV-2 PLpro/ubiquitin. NanoBRET based stable cell lines as presented here will be suitable for monitoring PPIs in other multiprotein complexes after various stimuli, mutations or small molecule administration and can be challenged with siRNA or CRISPR/Cas9 libraries to identify previously unrecognized regulators.

In the evolutionary battle between bacteria and mobile genetic elements, such as bacteriophage viruses and plasmids, bacteria have developed intricate defense systems. Among these, the CRISPR-Cas system has been extensively studied and harnessed as a revolutionary gene editing tool. However, while the biochemical process by which this microbial immune system acquires genetic CRISPR memory and immunity against invaders has been comprehensively examined, fundamental questions about the bacterial physiological state underlying how and when CRISPR memory is formed have only been partially explored. Naïve CRISPR adaptation is generally rare, but occurs more frequently when bacteria are challenged with replication-deficient phages. In such scenarios, bacteria are not under immediate threat and have ample time to adapt to the phage DNA, without risking cell death. Accordingly, slow growth caused by low temperatures, low aeration, or bacteriostatic antibiotics promotes CRISPR adaptation, possibly by allowing the Cas complexes more time to adapt before being outpaced. Persister cells are dormant antibiotic-tolerant subpopulations of cells with limited metabolic activity. When a mobile genetic element invades a persister cell, its replication is halted until the host cell resumes growth, providing an ideal opportunity for CRISPR adaptation. Here, we show that transiently dormant Escherichia coli persister cells acquire CRISPR immunity 10-fold more frequently than the general bacterial population. Thus, persister cells, in addition to being notoriously antibiotic tolerant, are primed for CRISPR-Cas adaptation and may be in a state of heightened immune capacity and evolution, securing the survival of the population.

ABSTRACT While GWAS have been successful in providing variant-to-trait associations for human complex diseases, functional dissection of the discovered loci has lagged behind. Here, we describe a variant-to-gene (V2G) mapping effort for Alzheimer’s disease (AD) to implicate causal variants and effector genes from the most recent AD GWAS meta-analyses (101 loci). We leveraged our genomics datasets comprising high-resolution promoter Capture C, ATAC-seq, and RNA-seq from brain-relevant cell types to fine-map AD GWAS variants, identifying 89 candidate causal SNPs and 69 effector genes. We then designed a single-cell CRISPRi screen to perturb candidate regulatory regions (n=74) and assess the transcriptional response in the human microglial cell line, HMC3. Our screen across ∼97,000 cells identified 19 regulatory regions and 19 effector genes. We then elected to functionally dissect our top hit, the TSPAN14 locus, and we show that an intronic region containing AD-associated SNPs rs7080009, rs1870138, and rs1870137 is a microglia-specific enhancer, with the AD risk haplotype increasing its activity. CRISPR precise genomic deletion of this region decreases TSPAN14 expression, alters specific cellular pathways including cell adhesion, and decreases secreted levels of pro-inflammatory cytokines IL-6 and IL-8, which are known biomarkers of aging and AD. Our work provides a systematic framework to map GWAS signals to their effector genes for AD and other brain-related disorders, and provides robust leads to follow up with in-depth functional investigations.

ABSTRACT Synthetic lethality (SL) is an extreme form of negative genetic interaction, where simultaneous disruption of two non-essential genes causes cell death. SL can be exploited to develop cancer therapies that target tumour cells with specific mutations, potentially limiting toxicity. Pooled combinatorial CRISPR screens, where two genes are simultaneously perturbed and the resulting impacts on fitness estimated, are now widely used for the identification of SL targets in cancer. Various scoring methods have been developed to infer SL genetic interactions from these screens, but there has been no systematic comparison of these approaches. Here, we performed a comprehensive analysis of 5 scoring methods for SL detection using 5 combinatorial CRISPR datasets. We assessed the performance of each algorithm on each screen dataset using two different benchmarks of paralog synthetic lethality. We find that no single method performs best across all screens but identify two methods that perform well across most datasets. GRAPHICAL ABSTRACT Figure 1. Graphical Abstract. Benchmarking Scoring Methods for Synthetic Lethality Detection from CRISPR screen data. Experimental setup for benchmarking experiments. Five different CRISPR double knockout (DKO) screens are scored for genetic interaction using 5 different scoring methods. The calculated scores are analysed using two different benchmarks (De Kegel Hits and Köferle Hits). Area under the receiver operating characteristic curve (AUROC) and Area under the precision recall curve (AUPR) for each scoring method on each dataset are calculated and compared.

The mouse model is by far the most widely used animal model in preclinical neuroscience, but translating findings to humans suffers from the lack of a formal framework establishing the correspondence between the mouse and the human brain. In this study, we build on the concept of common brain space, and on previous work embedding gene expression profiles, to bring the two species into alignment for comparative analysis. Using a variational autoencoder (VAE) combined with a latent classifier, we create a latent space that strikes a balance between abstract features related to reconstruction and features pertaining to regional segregation. This approach demonstrates improved cross-species homology and within-species locality compared to existing comparative models. In addition, we show that brain alterations in mouse disease models can be translated to humans, predicting patterns of brain changes in Alzheimer's and Parkinson's diseases. The flexibility and scalability of this approach offer a promising framework to bridge between more animal models, comparing quantitative imaging modalities, and disease phenotypes. This in turn will help advance our understanding of species similarities and differences, enhancing both fundamental translational neuroscience and disease modelling.

ABSTRACT RNA sequencing studies on human dorsal root ganglion (hDRG) from patients suffering from neuropathic pain show upregulation of OSM, linking this IL-6 family cytokine to pain disorders. In mice, however, OSM signaling causes itch behaviors through a direct effect on its cognate receptor expressed uniquely by pruriceptive sensory neurons. We hypothesized that an expansion in function of OSM-OSM receptor (OSMR) in sensory disorders in humans could be explained by species differences in receptor expression and signaling. Our in situ hybridization and immunohistochemical findings demonstrate broad expression of OSMR in DRG nociceptors and afferent fibers innervating the superficial and deep skin of humans. In patch-clamp electrophysiology, OSM directly activates human sensory neurons engaging MAPK signaling to promote action potential firing. Using CRISPR editing we show that OSM activation of MAPK signaling is dependent on OSMR and not LIFR in hDRG. Bulk, single-nuclei, and single-cell RNA-seq of OSM-treated hDRG cultures reveal expansive similarities in the transcriptomic signature observed in pain DRGs from neuropathic patients, indicating that OSM alone can orchestrate transcriptomic signatures associated with pain. We conclude that OSM-OSMR signaling via MAPKs is a critical signaling factor for DRG plasticity that may underlie neuropathic pain in patients.

Exonic enhancers (EEs) occupy an under-appreciated niche in gene regulation. By integrating transcription factor binding, chromatin accessibility, and high-throughput enhancer-reporter assays, we demonstrate that many protein-coding exons possess enhancer activity across species. These EEs exhibit characteristic epigenomic signatures, form long-range interactions with gene promoters, and can be altered by both nonsynonymous and synonymous variants. CRISPR–mediated inactivation demonstrated the involvement of EEs in the cis-regulation of host and distal gene expression. Through large-scale cancer genome analyses, we reveal that EE mutations correlate with dysregulated target-gene expression and clinical outcomes, highlighting their potential relevance in disease. Evolutionary comparisons show that EEs exhibit both strong sequence constraint and lineage-specific plasticity, suggesting that they serve ancient regulatory functions while also contributing to species divergence. Our findings redefine the landscape of functional elements by establishing EEs as a component of gene regulation, while revealing how coding regions can simultaneously fulfil both protein-coding and cis-regulatory roles.

Cells generate purine nucleotides through both de novo purine biosynthesis (DNPB) and purine salvage. Purine accumulation represses energetically costly DNPB through feedback inhibition of the enzymatic steps that produce the precursor phosphoribosylamine. Excessive DNPB is associated with human diseases including neurological dysfunction and hyperuricemia. However, the mechanisms explaining how cells balance DNPB and purine salvage are incompletely understood. Data from a genome-wide CRISPR loss-of-function screen and extensive stable isotope tracing identified Nudix hydrolase 5 (NUDT5) as a suppressor of DNPB during purine salvage. NUDT5 ablation allows DNPB to persist in the presence of either native purines or thiopurine drugs; this renders NUDT5-deficient cells insensitive to thiopurine treatment. Surprisingly, this regulation occurs independently of NUDT5’s known function in hydrolyzing ADP-ribose to AMP and ribose-5-phosphate. Rather, NUDT5 interacts with phosphoribosyl pyrophosphate amidotransferase (PPAT), the rate-limiting enzyme in DNPB that generates phosphoribosylamine. Upon induction of purine salvage, the PPAT-NUDT5 interaction is required to trigger disassembly of the purinosome, a cytosolic metabolon involved in efficient DNPB. Mutations that disrupt NUDT5’s interaction with PPAT but leave its catalytic activity intact permit excessive DNPB during purine salvage, inducing thiopurine resistance. Collectively, our findings identify NUDT5 as a regulator governing the balance between DNPB and purine salvage, underscoring its impact on nucleotide metabolism and efficacy of thiopurine treatment.

Objectives Candidozyma ( Candida ) auris is an emerging fungal pathogen of global concern that often exhibits multi-drug resistance. Over 90% of isolates are resistant to fluconazole. Of the six described clades of C. auris , Clade III has been found to be nearly universally fluconazole resistant and almost every Clade III isolate described carries a mutation in the gene encoding the fluconazole target sterol demethylase ( ERG11 ) leading to a VF125AL substitution and a mutation leading to a N647T substitution in the gene encoding Mrr1a, a transcriptional regulator of the Mdr1 transporter. Both mutations have been shown to contribute to fluconazole resistance in C. auris . Methods In the present study we introduced the Clade III MRR1A mutation into a Clade I background using CRISPR-Cas9 gene editing. In two Clade III clinical isolates we corrected the native MRR1A and ERG11 mutations to their wild-type sequences as well as disrupted MDR1 . Triazole susceptibilities and MDR1 gene expression were measured in all strains. Results Introduction: of the N647T substitution in a Clade I background confers a modest reduction in fluconazole and voriconazole susceptibility. Similarly, correction of MRR1A or disruption of MDR1 in each Clade III background resulted in a one-dilution decrease in fluconazole and voriconazole MIC while the ERG11 correction resulted in a three-dilution decrease in fluconazole and voriconazole MIC. Conclusions Our findings show that while the MRR1A mutation makes a modest contribution, the ERG11 mutation is responsible for most of the fluconazole resistance observed in Clade III isolates. We also show that while these mutations likewise affect voriconazole susceptibility, they have no effect on susceptibility to itraconazole, isavuconazole, or posaconazole suggesting the potential therapeutic utility of these antifungals for infections due to Clade III isolates of C. auris .

Interplay among gene-editing technologies, artificial intelligence (AI), and nanotechnology is revolutionizing personalized drug delivery with greater accuracy, efficiency, and individualized treatment regimens. This paper summarizes the development of lipid nanoparticles, vesicular drug carriers, and intelligent drug delivery systems and how these could improve drug bioavailability and targeted therapies. AI-based predictive models are revolutionizing drug discovery, formulation, and personalized treatment development to enable more efficient and personalized therapy.In addition, nanorobotics and magnetically triggered drug delivery are facilitating site-specific therapy with the specific significance in neurology and oncology. Further, CRISPR-based gene editing and artificial intelligence are facilitating precision of therapy with highly targeted nature's gene therapy. Advanced academe and industry technology are foreseeing the future for autonomous delivery systems and real-time monitoring facilitated by artificial intelligence to redefine precision medicine's era.This article highlights the revolutionizing capability of such inter-disciplinary advances and their ability to redefine contemporary therapeutics. Medicine in the future, supported by AI, nanotechnology, and gene editing, will be more effective, more specific, and patient-centric. Increased research and technological development will be the driving force for these advances to reach the clinic, and the outcome of therapy will become safer and more efficient.