Abstract CDKL5 deficiency disorder (CDD) is a severe X-linked neurodevelopmental condition characterized by early-onset epilepsy, intellectual disability, and motor dysfunction. Here, we present a dual-effector CRISPR-based epigenome editing platform that enables targeted reactivation of the silenced CDKL5 allele. Using a split dCas9 system compatible with AAV9 delivery, we achieved simultaneous transcriptional activation and DNA demethylation of the CDKL5 promoter in both murine and human models of disease. In heterozygous Cdkl5 E6del mice, intracerebroventricular delivery of the editor restored Cdkl5 protein expression, re-engaged downstream signaling, and rescued motor and cognitive deficits. In patient-derived neural stem cells and cortical organoids, CDKL5 reactivation normalized gene expression and restored neuronal network activity. These findings establish a scalable, mutation-independent strategy for treating CDD and highlight the therapeutic potential of epigenetic reprogramming for X-linked disorders.

Abstract While the combination of endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) improves progression-free survival (PFS) in HR+/HER2- metastatic breast cancer, resistance remains a major challenge. BRCA2 pathogenic variants have been linked to reduced PFS, potentially due to co-deletion of the neighboring RB1 gene on chromosome 13q. As RB1 is a key target of CDK4/6, its loss drives resistance. Using CRISPR/Cas9, we generated cell lines with single and combined BRCA2 and RB1 deletions. Loss of RB1 but not BRCA2 increased proliferation and conferred resistance to the CDK4/6i palbociclib and abemaciclib. Dual loss reduced proliferation but increased resistance to CDK4/6i in vitro . However, sensitivity to the PARP inhibitor olaparib was maintained. Finally, analysis of real-world clinical data revealed that RB1 mutations were more frequent in tumors exhibiting homologous recombination deficiency signatures and 13q loss. These genomic features were associated with shorter treatment duration on CDK4/6i plus ET. In conclusion, our findings suggest that RB1 loss, alone or with BRCA2 deletion, contributes to CDK4/6 inhibitor resistance and may help explain reduced efficacy in patients with BRCA2 mutations. Importantly, despite this resistance, sensitivity to PARP inhibition is retained, highlighting a potential therapeutic vulnerability in this molecular context.

Abstract Background: Small cell lung cancer (SCLC) is a highly aggressive malignancy characterized by rapid progression and the frequent emergence of resistance to standard chemotherapeutic agents such as cisplatin (DDP) and etoposide (VP16), resulting in poor clinical outcomes. Methods and Results: To elucidate mechanisms underlying chemoresistance, we conducted a genome-wide CRISPR/Cas9 knockout screen, which identified the histone demethylase KDM6B as a critical mediator of drug resistance in SCLC. Pharmacological inhibition of KDM6B using GSKJ1 markedly enhanced the sensitivity of drug-resistant SCLC cells to DDP and VP16. GSKJ1 treatment significantly suppressed cell proliferation and augmented chemotherapy-induced apoptosis, while exhibiting minimal cytotoxic effects when used as monotherapy. To explore the downstream regulatory pathways, we performed transcriptome analysis via RNA-seq followed by KEGG pathway enrichment analysis, which revealed that GSKJ1 treatment modulates key oncogenic signaling pathways. Integration of ChIP-seq data for H3K27me3 with transcriptomic profiles led to the identification of ERG3 as a potential downstream target. Protein interaction network analysis suggested that c-FOS is co-expressed with both KDM6B and ERG3. Co-immunoprecipitation (Co-IP) and Western blot (WB) assays confirmed the formation of a functional KDM6B/ERG3/c-FOS axis. Mechanistically, this axis regulates chemotherapy resistance by modulating apoptotic and ferroptotic pathways. Conclusion: Finally, in vivo experiments using patient-derived xenograft (PDX) models demonstrated that GSKJ1 effectively enhances the antitumor efficacy of chemotherapy in SCLC, providing compelling evidence for the clinical potential of targeting KDM6B to overcome chemoresistance.

Introduction In the kidney, epithelial cells of medullary collecting ducts (MCD) are exposed to a harsh microenvironment characterized by hyperosmolality, hypoxia, and oxidative stress. Still, under physiological conditions, they show no apparent signs of cellular injury. We hypothesized that the study of transcription factors (TFs) enriched in MCD cells might identify pathways of stress resilience. Methods We generated single-cell expression data from mouse kidneys and predicted in silico candidate TFs with MCD-enriched expression and activity. To investigate the transcriptomic effects of perturbing these candidate TFs, we devised a single-cell CRISPR interference (CRISPRi) screen in mouse inner medullary collecting duct (IMCD3) cells. Results We found that perturbation of interleukin enhancer-binding factor 2 (Ilf2), Krueppel-like factor 5 (Klf5), and JunB proto-oncogene (Junb), significantly impacted IMCD3 cells’ gene expression programs, including genes involved in proliferation, apoptosis, and stress-related intracellular signaling, highlighting these TFs as potential regulators of cell-specific resilience. We then focused on Ilf2, validated its impact on gene expression signatures by RNA sequencing, and identified a further role of Ilf2 in the splicing of genes involved in epithelial cellular dynamics and stress responses. Loss of Ilf2 function in IMCD3 cells resulted in nuclear atypia, reduced proliferative capacity, and increased cell death in response to osmotic stress. Ilf2 was upregulated in mouse kidneys during the repair phase following ischemia-reperfusion injury (IRI), as were Ilf2-regulated transcripts and splicing events, suggesting that Ilf2 might serve as a post-ischemic signal to facilitate epithelial stress resilience. Conclusions Our approach nominates novel pathways of cellular resilience in kidney tubular cells and highlights Ilf2 as a potential target for kidney protection. Translational Statement We identify Ilf2 as a transcriptional regulator promoting the stress resilience of renal epithelial cells. Our study provides mechanistic insights into the endogenous mechanisms that protect kidney epithelia from the adverse microenvironment of the renal medulla. Furthermore, Ilf2 activation might constitute a broader mechanism to preserve kidney epithelial integrity under injurious conditions.

Background: Neuroblastoma, the most common extracranial solid tumor in children, exhibits considerable clinical heterogeneity influenced by genetic predisposition. While genome-wide association studies (GWAS) in European populations have identified eight susceptibility loci, the genetic basis of neuroblastoma in East Asian populations remains poorly understood. Methods: We conducted the first GWAS in a Chinese cohort comprising 235 neuroblastoma patients and 3,100 controls, followed by multi-omics analyses of gene expression. The novel risk loci were further validated in an independent East Asian cohort (76 cases/269 controls). Functional characterization of a novel locus was carried out in neuroblastoma cell lines using CRISPR/Cas9-mediated deletion and overexpression assays to evaluate its regulatory effects on candidate genes. Findings: We replicated six of eight known loci including genome-wide significant associations at CASC15 (6p22.3; P = 1.55 E-09) and BARD1 (2q35;P = 3.44E-07), and identified 11 novel risk loci. These novel associations implicate genes involved in DNA repair (MUTYH at 1p34.1), neurodevelopment (BASP1 at 4q13.2 and SLC22A4/SLC22A5 at 5q31.1), and immune regulation (HLA at 6p21 and IDO1/IDO2 at 5q31.1). Multi-omics integration revealed that lead variants modulate gene expression (cis-eQTLs) and DNA methylation (mQTLs) in neural crest-derived tissues and immune cells. Two loci (rs2631372 at 5q31.1: P= 0.045; rs2956095 at 11p13: P= 0.027) showed consistent associations in the replication cohort. Functional studies demonstrated that deletion of the 5q31.1 risk interval reduced expression of SLC22A4, SLC22A5, and LOC553103, while their overexpression promoted neuroblastoma cell proliferation. Interpretation: These findings highlight both shared and population-specific genetic contributions to neuroblastoma susceptibility, underscoring the importance of diversifying GWAS efforts to advance ancestry-informed risk assessment and therapeutic strategies.

Resistance to endocrine therapy (ET) remains a major clinical challenge in the treatment of estrogen receptor–positive (ER⁺) breast cancer, underscoring the need for novel therapeutic targets. To identify genetic drivers of ET resistance, we conducted an in vivo genome-wide CRISPR-Cas9 screen in MCF7 cells implanted into ovariectomized nude mice under estrogen-deprived conditions. NFKB1 emerged as a top candidate whose loss promoted estrogen-independent tumor growth and recurrence. Functional studies confirmed that NFKB1 deficiency enhanced tumorigenicity and conferred resistance to tamoxifen and fulvestrant both in vitro and in vivo. Mechanistically, transcriptomic and biochemical analyses revealed that NFKB1 loss activated canonical NF-κB signaling, leading to inflammatory gene induction and hyperactivation of ER signaling. Importantly, pharmacologic inhibition of NF-κB signaling restored ET sensitivity in NFKB1-deficient cells. Clinically, NFKB1 downregulation was enriched in ER⁺ breast tumors and associated with poor patient outcomes. Collectively, these findings establish NFKB1 as a key suppressor of ET resistance, uncover a mechanistic link between inflammation and ER reactivation, and highlight NF-κB signaling as a therapeutic vulnerability in NFKB1-deficient ER⁺ breast cancer.

ABSTRACT Antimicrobial resistance is a major global health threat, with disproportionate impact in regions with limited diagnostic infrastructure. To address this challenge, we developed BADLOCK (Bacterial and AMR Detection by SHERLOCK), a rapid, low-cost molecular diagnostic platform for direct detection of bacterial pathogens and resistance genes from clinical samples. BADLOCK operates as a one-pot CRISPR-Cas13a reaction capable of detecting nine bacterial species and four major resistance genes directly from positive blood culture. It requires only a heat block and supports both fluorescence and paper-based lateral flow readouts. We validated BADLOCK on a prospectively collected clinical cohort of 194 blood culture specimens, supplemented with 69 mock samples generated from banked isolates enriched for targeted resistance genes. Across all cohorts, we conducted 2,224 individual reactions, achieving 97.6% accuracy (2,171/2,224) at the reaction level. At the assay level, 89.5% (274/306) showed perfect or partial concordance with gold-standard species and resistance gene detection, including 255 assays with perfect concordance and 19 with partial concordance (correct detection of at least one pathogen). This included an evaluation of BADLOCK as a potential culture-free diagnostic for urinary tract infections (UTIs), achieving 98.0% reaction-level accuracy. At the assay level, 90.7% (41/43) were perfectly concordant with gold-standard detection of both species and resistance genes, with 2 additional assays showing partial concordance. To our knowledge, this represents the first demonstration of the CRISPR-Cas13a diagnostic platform on clinical bloodstream infections to date and supports BADLOCK’s potential as a practical and scalable solution for rapid pathogen and resistance gene detection in resource-constrained settings.

Recognition of protospacer adjacent motifs (PAMs) is crucial for target site recognition by CRISPR–Cas systems. In genome editing applications, the requirement for specific PAM sequences at the target locus imposes substantial constraints, driving efforts to search for novel Cas9 orthologs with extended or alternative PAM compatibilities. Here, we present CRISPR-PAMdb, a comprehensive and publicly accessible database compiling Cas9 protein sequences from 3.8 million bacterial and archaeal genomes and PAM profiles from 7.4 million phage and plasmid sequences. Through spacer–protospacer alignment, we inferred consensus PAM preferences for 8,003 unique Cas9 clusters. To extend PAM discovery beyond traditional alignment-based approaches, we developed CICERO, a machine learning model predicting PAM preferences directly from Cas9 protein sequences. Built on the ESM2 protein language model and trained on the CRISPR–PAMdb database, CICERO achieved an average accuracy of 0.68 on test data and 0.75 on experimentally validated Cas9 orthologs. For Cas9 clusters where alignment-based predictions were infeasible, CICERO generated PAM profiles for an additional 50,308 Cas9 proteins, including 17,453 high-confidence predictions with accuracies above 0.86. CRISPR–PAMdb, alongside CICERO models, enables large-scale exploration of PAM diversity across Cas9 proteins, accelerating design of next-generation CRISPR-Cas9 tools for precise genome engineering.

Summary In most legume-rhizobium symbioses, rhizobial colonization occurs through host-derived intracellular infection threads, which enable recruitment of compatible rhizobia while presumably modulating the host immune system to prevent rejection. To investigate how legumes regulate immune responses through post-translational mechanisms during the infection, we focused on Cyclophilin A (CyPA), a peptidyl-prolyl cis/trans isomerase. The model legume Lotus japonicus encodes three canonical CyPA genes. Among them, LjCyPA1 was characterized through CRISPR/Cas9-mediated knockout analysis and shown to be important for normal intracellular infection of compatible rhizobia. A gain-of-function LjCyPA1 variant in a soybean cultivar was able to promote symbiosis with not only compatible but also incompatible rhizobia. Structural modeling followed by genetic analysis demonstrated a functional interaction between LjCyPA1 and the immune hub protein LjRIN4. The cis conformation of LjRIN4 promoted intracellular rhizobial infection, while the trans conformation suppressed it. LjCyPA1 acted with the rhizobial type III secretion system (T3SS) which exhibited a cooperative role between host and symbiont in facilitating infection. Phylogenomic analysis showed that conservation of the CyPA1 orthologue is correlated with the trait of intracellular infection in legumes. Our results contribute to the understanding of how legumes accept symbiotic partners while balancing immune responses.

Trehalose-6-phosphate (Tre6P) is the intermediate in the two-step pathway of trehalose biosynthesis mediated by Tre6P-synthases (TPSs) and Tre6P-phosphatases (TPPs). Plants harbor small families of TPS and TPP genes, however most plant TPSs lack enzymatic activity, suggesting they have regulatory functions. The classical mutant ramosa3 (ra3) increases inflorescence branching in maize, and RA3 encodes a catalytic TPP. We found that RA3 interacts with maize ZmTPS1, a non-catalytic TPS. Mutants in ZmTPS1 and its close paralog ZmTPS12 enhance ra3 phenotypes, suggesting their physical interaction is biologically significant. ZmTPS1 also interacts with the two catalytically active maize TPSs, ZmTPS11 and ZmTPS14, however zmtps11;zmtps14 double mutants fail to complete embryogenesis, suggesting that they are essential, as in arabidopsis. Interestingly, the non-catalytic ZmTPS1 protein stimulated the coupled activity of RA3 and ZmTPS14, suggesting that RA3, ZmTPS1, and ZmTPS14 form a complex, and we confirmed this by expressing and purifying the three proteins and by Alphafold predictions. Our results suggest that non-catalytic TPSs form a complex with catalytic TPSs and TPPs to stimulate catalytic activity and regulate plant development.

The basic helix-loop helix transcription factor Twist plays diverse roles in mesodermal development across bilaterians, but its function in cnidarians remains unclear. Here, we investigate the role of Twist in tentacle morphogenesis and tissue homeostasis in the sea anemone Nematostella vectensis . Using a CRISPR/Cas9 generated knockout, we show that twist mutants exhibit impaired secondary tentacle formation, reduced proliferation in budding tentacles. Cross-sections reveal that mutants also lack micronemes, which are incomplete mesenteries that demarcate tentacle boundaries-suggesting defects in spatial patterning. We demonstrate that twist expression is regulated by Wnt, BMP, and Notch signalling but is independent of MAPK and Hedgehog pathways. Loss of Twist disrupts expression of mesodermal transcription factors paraxis and tbx15 and perturbs the TOR-FGF signalling feedback loop necessary for normal tentacle growth. In addition to the impaired tentacle formation phenotype, juvenile or adult mutants develop epithelial neoplasms at the level of the pharynx, with tentacle-like molecular and morphological profiles, indicating a role for Twist in maintaining tissue homeostasis at the oral pole. Together, our findings reveal that Twist integrates major signalling pathways to regulate secondary tentacle patterning and maintain spatial tissue organisation in the diploblastic Nematostella vectensis .

Gene family expansions are critical for functional diversification, yet paralog contributions to metabolic pathways are often unclear. In Caenorhabditis, the expanded O-acyltransferase (OAC) family, enzymes that transfer acyl groups to hydroxylated substrates, remains poorly characterized despite having been implicated in lipid metabolism. Using CRISPR-Cas9 mutagenesis, behavioral assays, gas chromatographic-mass spectral (GC-MS) analyses, and metabolomics, we systematically analyzed 59 OAC-family protein-coding genes to define their roles in regulating signaling molecules. We found that four adjacent paralogs (oac-13, oac-16, oac-25, and oac-28) on chromosome I are required for synthesizing volatile sex pheromones (VSPs), airborne signals critical for male mate-searching. Specifically, oac-13 and oac-16 are necessary for producing both major pheromone components, while the identical tandem paralogs oac-25 and oac-28 regulate the production of the later-eluting component in gas chromatography. Disruption of these genes reduced production of key pheromone components and impaired male attraction. Metabolomics revealed that oac-16 and other OACs also modulate synthesis and secretion of non-volatile ascaroside pheromones, indicating dual roles in chemical signaling. This work uncovers functional specialization within an expanded gene family, illustrating how redundancy and divergence enable adaptive evolution of communication systems.

Polyglutamine (polyQ) diseases, including Huntington's disease and several spinocerebellar ataxias, are caused by abnormally expanded CAG nucleotide repeats, which encode aggregation-prone polyQ tracts. Substantial prior evidence supports a pathogenic role for polyQ protein misfolding and aggregation, with molecular chaperones showing promise in suppressing disease phenotypes in cellular and animal models. In this study, we developed a FRET-based reporter system that models polyQ aggregation in human cells and used it to perform a high-throughput CRISPR interference screen targeting all known molecular chaperones. This screen identified as a strong suppressor of polyQ aggregation the Hsp40 co-chaperone DNAJC7, which has previously been shown to modify aggregation of other disease proteins (tau and TDP-43) and has mutations causative for amyotrophic lateral sclerosis. We validated this phenotype and further established a physical interaction between DNAJC7 and polyQ-expanded protein. In contrast, DNAJC7 did not modify aggregation of polyglycine (polyG) in a FRET-based model of neuronal intranuclear inclusion disease. In addition to establishing new inducible, scalable cellular models for polyQ and polyG aggregation, this work expands the role of DNAJC7 in regulating folding of disease-associated proteins.

Transcriptional regulation is tightly linked to chromatin organization, with H3K4me3 commonly marking both active and bivalent promoters. In embryonic stem cells (ESC), MLL2 is essential for H3K4me3 deposition at bivalent promoters, which has been proposed to facilitate the induction of major developmental genes during pluripotent cell differentiation. However, prior studies point to a functional discrepancy between the loss of H3K4me3 at bivalent promoters and the largely unaltered transcription of major developmental genes in Mll2 -/- cells. In this study, we investigated MLL2-dependent gene regulation in mouse ESC and during their differentiation. Contrary to the prevailing view, we show that MLL2’s primary role is not to oppose Polycomb-mediated repression at the bivalent promoters of developmental genes. Instead, we identify a previously unrecognized regulatory function for MLL2 at the CG-rich 5’ untranslated regions (5’UTR) of evolutionarily young LINE-1 (L1) transposable elements (TE). We found that MLL2 binds to the 5’UTR of L1 elements and is critical for maintaining their active state (H3K4me3 and H3K27ac), while preventing the accumulation of repressive H3K9me3. Using both global genomic approaches (i.e. RNA-seq, ChIP-seq and Micro-C) as well as targeted L1 deletions, we demonstrate that these MLL2-bound L1 elements act as enhancers, modulating the expression of neighboring genes in ESC and, more prominently, during differentiation. Together, our findings illuminate novel aspects of MLL2 regulatory function during early developmental transitions and highlight the emerging role of TE as key components of long-range gene expression control.

Cardiac development is characterized by a complex series of molecular, cytoskeletal and electrophysiological changes that guarantee the proper functioning of adult cardiomyocytes (CMs). These changes are defined by cell-type-specific transcriptional rewiring of progenitor cells to form CMs, and are regulated by various epigenetic elements, such as long noncoding RNAs (lncRNAs). LncRNAs are versatile epigenetic regulators as they may act in cis or in trans to orchestrate important gene programs during cardiac development and may concurrently encode micropeptides. LIPTER is one such lncRNA, previously shown to regulate lipid droplet transport in cardiomyocytes and thus an important regulator of cardiomyocyte metabolism. Here we show that LIPTER also plays a role in the cytoskeletal maturation of CMs, as loss of LIPTER leads to persistent expression of fetal genes, changes in chromatin accessibility, disorganized sarcomeres and impaired calcium homeostasis in CMs. Furthermore, we have identified a cardiomyocyte-specific regulatory enhancer that regulates the expression of LIPTER in CMs. CRISPR-mediated inhibition of this enhancer led to reduced LIPTER expression in CMs and increased expression of fetal genes. This CM-specific enhancer could therefore be manipulated to control the expression of LIPTER for therapeutic benefit. In summary, we have unravelled a novel role of LIPTER in CMs cytoskeletal maturation and have identified a CM-specific enhancer for LIPTER.

Discovery of key growth drivers that can be targeted for therapy is a central goal in cancer research. While high-throughput CRISPR screens have revolutionized our ability to identify gene dependencies in cancer, most large-scale screens are conducted in two-dimensional (2D) culture systems that fail to recapitulate tumor organization and behavior. To uncover architecture-dependent vulnerabilities in breast cancer, we performed parallel CRISPR interference (CRISPRi) screens in 2D and three-dimensional (3D) cultures of MCF7 cells, an estrogen receptor-positive (ER+) breast cancer model representative of a high risk of relapse, luminal subtype. Knockdown of IFNAR2 and TYK2 conferred a growth advantage in 3D cultures, implicating type I interferon signaling as a tumor-intrinsic suppressor of proliferation in 3D spheroids. Transcriptomic and functional analyses demonstrated that type I IFN signaling is endogenously activated in 3D spheroids via RIG-I-mediated sensing of cytosolic double-stranded RNA, leading to TBK1 activation and induction of interferon-stimulated genes (ISGs). This tumor-intrinsic IFN response slowed proliferation in 3D culture, independent of exogenous stimuli or the presence of immune cells. Analysis of bulk, single-cell, and spatial transcriptomic datasets from breast cancer patients revealed that a subset of tumors exhibit elevated IFN signaling in cancer cells, including in immune-depleted tumor cores, consistent with a tumor-intrinsic IFN signature. Our findings uncover an IFN-mediated growth-suppressive program shaped by 3D tumor architecture, and contribute towards a better understanding of the role of tumor-intrinsic IFN activity.

Background Tegumentary leishmaniasis is a parasitic disease endemic in the Americas. Its clinical management and control rely on early and accurate diagnosis and adequate treatment. PCR-based molecular diagnostics offer high sensitivity and specificity over microscopy or culture but are less accessible in low-resource settings. New molecular tools for detecting Leishmania infections are needed in rural endemic regions. A promising tool harnessing CRISPR-Cas technology enables highly specific and sensitive detection of nucleic acid targets, offering an exciting potential for portable molecular diagnostics. Previously, we developed CRISPR-Cas12a-based assays coupled to PCR preamplification for Leishmania detection. Here, we adapted our assays, which target the 18S rDNA and kinetoplast DNA (kDNA) minicircles, by replacing PCR with loop-mediated isothermal amplification (LAMP). Methodology/Principal Findings LAMP-CRISPR assays were optimized for fluorescence-based and lateral flow readouts. The assays could detect as low as 0.2 genome equivalents per reaction using L. braziliensis M2904 strain genomic DNA. The kDNA assay reliably detected all tested species of the Leishmania ( Viannia ) subgenus, while the 18S assay showed pan- Leishmania detection capability. There was no cross-reactivity with other protozoan ( Trypanosoma cruzi and Plasmodium falciparum ) and bacterial ( Mycobacterium tuberculosis ) pathogen DNA, or with human DNA. When applied to 90 clinical samples (skin lesions) from the Cusco region of Peru and compared to kDNA real-time PCR, LAMP-CRISPR assays with a fluorescence readout achieved a sensitivity of 90.9% for kDNA and 72.7% for 18S rDNA, both with 100% specificity. Overall, lateral flow strip results agreed with fluorescence-based detection in 18 tested samples, with one discrepancy observed in the 18S assay associated with low parasite load. Conclusions/Significance These new assays, being amenable to further simplification and optimization for their adoption in low-resource settings, hold promise as a new generation of accurate molecular tools for leishmaniasis diagnosis and surveillance, supporting One Health strategies for disease control. Author Summary Tegumentary leishmaniasis affects poverty-related populations in the Americas and encompasses skin and mucosal lesions that can cause disfigurement and social stigma. The disease is caused by several species of the protozoan parasite Leishmania. PCR-based molecular diagnostics are currently the most sensitive and specific diagnostic tools. Yet, these require specialized infrastructure and trained personnel that are not readily available in low-resource settings. New tools are required to meet the diagnostic needs in rural endemic areas. A promising tool leveraging CRISPR-Cas technology enables cost-effective, in vitro nucleic acid detection, paving the way for diagnostic solutions that could be made available to patients at, or near, the point of care. Here, we harnessed the CRISPR-Cas12a system combined with loop-mediated isothermal amplification (LAMP) to develop assays capable of detecting multiple Leishmania species of medical importance. Our assays employ multi-copy targets widely used in molecular diagnostics: the 18S rDNA for pan-Leishmania detection and a kDNA minicircle region conserved among L. (Viannia) species. Results can be read with either fluorescence detection or lateral flow strips. Both assays showed satisfying performance in both analytical validation and clinical sample testing under laboratory conditions. These new tools show promise to improve diagnosis and surveillance of leishmaniasis.

Multiplexed methods for nucleic acid detection are immensely challenging to deploy outside of laboratory settings. Conversely, field-deployable methods are limited to low levels of multiplexing. During the COVID-19 pandemic, we developed Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a sensitive and deployable CRISPR-based technology for nucleic acid detection. Here, we introduce microfluidic SHINE (mSHINE) which enables >100-plex nucleic acid detection using a highly portable microfluidic manifold. The manifold directs a diluted sample into individual reaction chambers, each of which contains lyophilized SHINE reagents and a microscopic stir bar or bead for mixing. Samples can be loaded using a syringe by hand, greatly simplifying the testing process. A subsequent sealing step allows for >100 SHINE reactions to proceed independently and in parallel. We demonstrate that mSHINE has equal sensitivity to SHINE, allowing for highly multiplexed pathogen detection in ≤ 1 hour. In addition, mSHINE can detect single-nucleotide variants, including mutations associated with drug susceptibility. mSHINE shifts the paradigm of laboratory-based multiplexed nucleic acid testing, greatly benefiting patients and public health.

ABSTRACT Pseudomonas aeruginosa is a metabolically versatile opportunistic human pathogen. It causes acute and chronic infections and is notorious for its multidrug resistance and tolerance. To systematically uncover genetic vulnerabilities that could be exploited as therapeutic targets, we present a portable high-density CRISPR interference (CRISPRi) library comprising >80’000 single-guide RNAs (sgRNAs) targeting virtually all annotated coding sequences and intergenic regions of P. aeruginosa PAO1. This library was used to assess the genome-wide fitness landscapes under different growth conditions, uncovering gain- and loss- of-function phenotypes for more than a thousand genes upon depletion. Many of the phenotypes are likely caused by hypomorphic (partial loss-of-function) alleles that would not be easily accessible by traditional transposon sequencing (Tn-Seq). Focusing on central carbon metabolism, we reveal two glyceraldehyde-3-phosphate dehydrogenases as central, non-redundant nodes in glycolytic and gluconeogenic growth conditions that might be promising targets to redirect carbon flux away from metabolically persistent states associated with chronic infections. More generally, our comprehensive sgRNA libraries are a valuable resource to access genome-wide quantitative phenotypes through CRISPRi beyond the binary phenotypes offered by Tn-Seq.

ABSTRACT Acral melanoma (AM) is an aggressive melanoma subtype with limited therapeutic options and poor outcomes. In non-European descent and admixed populations, like those residing in Latin America, AM accounts for a significant proportion of cutaneous melanoma cases. Here, we performed comprehensive genomic and functional profiling of AM from a uniquely diverse Brazilian cohort. Whole-exome and transcriptome sequencing revealed low mutation burden and predominance of copy number alterations, including high-amplitude focal amplifications termed hailstorms. These hailstorms frequently affected chromosomes 11, 5 and 22 and key oncogenes such as CCND1 , GAB2 , CDK4 , and TERT . The presence of hailstorms in the long arms of chromosomes 11 and 22 was associated with higher focal copy number burden and loss of DNA damage response genes ( ATM , CHEK1 ), suggesting a permissive genomic environment driving structural instability. To explore the unique genomic context of AM, we established a comprehensive collection of patient-derived xenograft (AM-PDX) models that faithfully retain the histopathological and genomic features of the original tumours. Functional exploration of AM-specific vulnerabilities through pharmacological and CRISPR/Cas9 knockout screenings identified strong sensitivity to targeting MAPK, CDK4/6, MDM2, and WEE1 pathways. Notably, the pan-RAS(ON) inhibitor RMC-7977 effectively reduced viability in NRAS -, KRAS -, and KIT -mutant AM cell lines. Finally, CRISPR screens revealed dependencies selectively essential in AM, including CRKL and SF3B4 , highlighting previously unrecognized vulnerabilities. Our findings emphasize the distinct biology of AM compared to other subtypes of melanoma, provide a valuable resource of models reflective of Latin American ancestry, and identify potential drivers and therapeutic targets.

Closely related species often exhibit distinct morphologies that can contribute to species-specific adaptations and reproductive isolation. One example are Lepidopteran caterpillar appendages, such as the “caudal horn” of Bombycoidea moths, which have evolved substantial morphological diversity among species in this group. Using interspecific crosses, we identify the genetic basis of the caudal horn size difference between Bombyx mori and its closest relative B. mandarina . The three largest of eight QTL account for one third the mean horn length difference between the species. The largest of these, on chromosome 4, encompasses a conserved Wnt -family gene cluster, key upstream regulators that are well-known for their roles in morphological diversification in animals. Using allele-specific expression analysis and CRISPR/Cas9 knockouts, we show that tissue-specific cis-regulatory changes to Wnt1 and Wnt6 contribute to the species difference in caudal horn size. This kind of modularity enables highly pleiotropic genes, including key upstream growth regulators, to contribute to the evolution of morphological traits without causing widespread deleterious effects. Significance This study explores the genetic basis of a distinct morphological trait that varies between two closely related moth species, providing insights into the evolution of morphological diversity. By identifying cis-regulatory changes in two Wnt -family genes as major contributors, this work underscores the importance of developmental gene regulatory networks in shaping species-specific traits. The findings illustrate how even small modifications in major upstream regulator genes can drive significant phenotypic variation, revealing how genetic changes in key growth regulators fuel the diversification of form and function. These results advance our understanding of the mechanisms behind the evolution of complex morphological traits.

Abstract Background The behaviour of complex biological systems emerges from the coordinated activity of networked molecular components. In this context, gene regulatory networks (aka gene coexpression networks) offer insights into the regulation of gene expression programs. In cancer, aberrant gene expression underlies molecular and clinical features, and identifying key networked transcriptional regulators may enable targeted therapeutic interventions. However, computationally inferred regulatory nodes have so far hardly been experimentally validated. Results Here we combined gene expression network analysis with gene perturbation experiments to test whether computationally identified hub genes act as upstream regulators of their coexpression modules in breast cancer. To better capture the context-dependent nature of gene regulation and minimize confounding effects due to heterogeneity, we also constructed subtype-specific networks. Using the METABRIC transcriptomic dataset of primary breast tumours, we identified clinically-informative gene modules in the highly aggressive basal-like subtype. Candidate regulatory hubs were prioritized based on network centrality, and their functional relevance was assessed both in silico and in vitro . CRISPR-mediated knockout of selected hub genes resulted in coordinated down-regulation of module genes and impaired cellular functions, demonstrating causal links between hub gene function, module expression and phenotypic outcome. Moreover, we observed a significant correlation between the transcriptional impact of each knockout and its functional effects—highlighting the biological relevance of coexpression modules and supporting the hypothesis that their structure reflects functional dependencies. Conclusions To our knowledge, this is the first study to functionally validate clinically relevant hub genes, providing direct support for the predictive power of coexpression-based network models.

Abstract Midbrain dopaminergic neurons (mDA) are selectively lost in Parkinson’s disease (PD), driving sustained efforts to generate bona fide mDA neurons from human-induced pluripotent stem cells (iPSCs) for replacement therapy. While morphogen gradients and transcription factors have been extensively studied, extracellular regulators remain largely overlooked. Here, we identify the heparan sulfate-modifying enzymes SULF1 and SULF2 as essential for establishing mDA neuron identity in vitro. Using CRISPR/Cas9-engineered iPSCs, we show that loss of SULF1/2 increases 6-O-sulfation of heparan sulfate chains and disrupts anterior-posterior and dorsoventral patterning in cells exposed to a midbrain differentiation protocol. Double-knockout cells fail to acquire midbrain fate and instead adopt caudal and neural crest-like identities, as revealed by single-nucleus RNA sequencing. Mechanistically, we find enhanced FGF signaling and demonstrate that FGF inhibition redirects cells toward midbrain progenitors, without fully restoring ventral identity. These findings establish a critical role for SULF1/2 in human mDA neuron development and uncover a previously unrecognized layer of extracellular control over neuronal patterning, opening for novel strategies to refine differentiation protocols for PD and beyond.

White Spot Syndrome Virus (WSSV) is one of the most devastating viral pathogens affecting shrimp, causing severe economic losses to the global farmed shrimp trade. The globalization of live shrimp trade and water-borne transmission have facilitated the rapid spread of WSSV across major shrimp-producing countries since its initial emergence. The present review gives an updated account of WSSV biology, pathology, transmission dynamics, and recent developments in control measures. The virus, a double-stranded DNA virus of the Nimaviridae family, utilizes advanced immune evasion strategies, resulting in severe mortality. Shrimp lack adaptive immunity and hence rely predominantly on innate immunity, which is insufficient to mount an effective response against severe infections. Traditional disease control measures, such as augmented biosecurity, selective breeding, and immunostimulants, have, despite extensive research, achieved only limited success. New biotechnological tools, such as RNA interference, CRISPR-Cas gene editing, and nanotechnology, offer tremendous potential for disease mitigation. In parallel, the development of DNA and RNA vaccines targeting WSSV structural proteins, such as VP28, holds significant promise for stimulating the shrimp immune system. This review highlights the urgent need for a convergent approach to sustainable disease management in global shrimp aquaculture, with interdisciplinarity playing a pivotal role in shaping the future of WSSV control.

CRISPR/Cas9 based genome editing employing Homology Directed Repair (HDR) from template vector sequences is a widely used technique to enable precise insertions, deletions or modifications to genes. Here, we describe an undesired and highly frequent editing event when using conventional CRISPR/Cas9 plus HDR methods for Drosophila melanogaster germline genome editing. We find that the template vector employed for HDR repair unwantedly and commonly inserts into the genome. We observe this deviation from the desired edit at multiple genomic locations, with different HDR vectors and with multiple genome editing designs. To avoid these events, we have generated a novel HDR template vector that enables animals with these undesired insertions to be identified and excluded. Our results suggest that HDR based genome edited animals must be carefully screened for unwanted vector template genomic integration in order to avoid misleading interpretations of genome editing outcomes.