Biological rhythms not only coordinate cellular activity with external signals, but may also enable internal coordination. The metabolic cycle in budding yeast is perhaps the most well-studied example. Historically researchers have investigated this cycle in populations growing in chemostats, but more recently time-lapse microscopy has revealed single-cell oscillations in the redox state of enzyme cofactors and in ATP levels. How to relate the results of these two types of assays is however unclear. Here we report single-cell rhythms too in intracellular pH and show that oscillations in the redox state of flavin molecules occur in auxotrophic and prototrophic strains, in nutrients favouring respiration or fermentation, and in deletion mutants for which oscillations in chemostats are either unobservable or disrupted. To explain the pervasiveness of these rhythms, we postulate that cells generate oscillations to alleviate a proteome constraint – amino acids cells use for one class of enzymes are unavailable for others. Using flux balance analysis with an enzyme-constrained genome-scale metabolic model, we show that, with a finite proteome, sequential synthesis of biomass components typically generates a shorter doubling time than synthesising components in parallel. Our results suggest that the metabolic cycle drives growth and is potentially widespread because all cells grow within a proteome constraint.

Disruption of parvalbumin positive (PVALB+) cortical interneurons is implicated in the pathogenesis of schizophrenia. However, how these defects emerge during brain development remains poorly understood. The protracted maturation of these cells during postnatal life has made their derivation from human pluripotent stem cells (hPSCs) extremely difficult, precluding hPSC-based disease modeling of their role in neuropsychiatric disease. Here we present a cortical assembloid system that supports the development of PVALB+ cortical interneurons which match the molecular profiles of primary PVALB+ interneurons and display their distinctive electrophysiological features. Further, we characterized cortical interneuron development in a series of CRISPR-generated isogenic structural variants associated with schizophrenia and identified variant-specific phenotypes affecting cortical interneuron migration and the molecular profile of PVALB+ cortical interneurons. These findings offer plausible mechanisms on how the disruption of cortical interneuron development may impact schizophrenia risk and provide the first human experimental platform to study of PVALB+ cortical interneurons.

ABSTRACT The difficulty of accurately identifying Candida auris and the high resistance rates presented have increased the concern in the healthcare setting. Due to this, the aim of this study was to analyse the fungal response to oxidative stress. To achieve this goal, gene and protein expression were examined using qPCR and two-dimensional electrophoresis, respectively, peroxiredoxin Tsa1b being discovered to be overexpressed under oxidative stress. Besides, its antigenicity was also confirmed by western blotting. Subsequently, the significance of Tsa1b was next investigated by creating and characterizing the C. auris ΔTSA1B and C. auris ΔTSA1B::TSA1B strains using CRISPR-Cas9. The findings demonstrated that the ΔTSA1B strain was more susceptible to oxidative and cell wall stressors than the wild-type strain, which was consistent with an increase in the cell wall β-glucan amounts when grown in the presence of oxidative stress. Furthermore, the ΔTSA1B strain was also more vulnerable to the presence of dendritic cells and bone marrow-derived macrophages. Finally, in vivo infections performed in Galleria mellonella and mice showed a slower progression of the disease in those animals infected with the mutant strain. In conclusion, the peroxiredoxin Tsa1b has been identified as an important protein for the C. auris response to oxidative stress and as a virulence factor, allowing for a more thorough knowledge of the pathobiology of this yeast. This study points out the potential that this protein may have for the development of new diagnostic and therapeutic approaches. HIGHLIGHTS Several metabolic proteins are implicated in C. auris response to oxidative stress C. auris response to oxidative stress is influenced by the Tsa1b peroxiredoxin Lack of Tsa1b generates more susceptibility to stresses and an altered cell wall C. auris Tsa1b is involved in the fungal interaction with host immune cells The Tsa1b of C. auris contributes to the progression of the infection in vivo

ABSTRACT CRISPR-Cas technologies have revolutionized life sciences by enabling programmable genome editing across diverse organisms. Achieving dynamic and precise control over CRISPR-Cas activity with exogenous triggers, such as light or chemical ligands, remains an important need. Existing tools for CRISPR-Cas control are often limited to specific Cas orthologs or selected applications, restricting their versatility. Anti-CRISPR (Acr) proteins, natural inhibitors of CRISPR-Cas systems, provide a flexible regulatory layer but are constitutively active in their native forms. In this study, we built on our previously reported concept for optogenetic CRISPR-Cas control with engineered, light-switchable anti-CRISPR proteins and expanded it from ortholog-specific Acrs towards AcrIIA5 and AcrVA1, broad-spectrum inhibitors of CRISPR-Cas9 and -Cas12a, respectively. We then conceived and implemented a novel, chemogenetic anti-CRISPR platform based on engineered, circularly permuted ligand receptor domains of human origin, that together respond to six different, clinically-relevant drugs. The resulting toolbox achieves both optogenetic and chemogenetic control of genome editing in human cells with a wide range of CRISPR-Cas effectors, including type II-A and -C CRISPR-Cas9s, and -Cas12a. In sum, this work establishes a versatile platform for multidimensional control of CRISPR-Cas systems, with immediate applications in basic research and biotechnology and potential for therapeutic use in the future.

ABSTRACT Here, we present M2D2, a two-stage machine learning (ML) pipeline that identifies promising antimicrobial drug combinations, which are crucial for combating drug resistance. M2D2 addresses key challenges in drug combination discovery by predicting drug synergies using computationally generated drug-protein interaction data, thereby circumventing the need for expensive omics data. The model improves the accuracy of drug target identification using high-throughput experimental and computational methods via feedback between ML stages. M2D2’s transparent framework provides mechanistic insights into drug interactions and was benchmarked against chemogenomics, transcriptomics, and metabolomics datasets. We experimentally validated M2D2 using high-throughput screening of 946 combinations of Food and Drug Administration (FDA)- approved drugs and antibiotics against Escherichia coli . We discovered synergy between a cerebrovascular drug and a widely used penicillin antibiotic and validated predicted mechanisms of action using genome-wide CRISPR inhibition screens. M2D2 offers a transparent ML tool for rapidly designing combination therapies and guides repurposing efforts while providing mechanistic insights.

Abstract Staphylococcus aureus Cas9 (SaCas9), which is smaller than the widely-used Streptococcus pyogenes Cas9 (SpCas9), has been harnessed for gene therapy using an adeno-associated virus vector. However, SaCas9 requires an NNGRRT (where N is any nucleotide and R is A or G) protospacer adjacent motif (PAM) for target DNA recognition, thereby restricting the targeting range. In addition, the nuclease activation mechanism of SaCas9 remains elusive. Here, we rationally engineered a SaCas9 variant (eSaCas9-NNG) with an expanded target scope and reduced off-target activity. eSaCas9-NNG induced indels and base conversions at endogenous sites bearing NNG PAMs in human cells and mice. We further determined the cryo-electron microscopy structures of eSaCas9-NNG in four sequential states, PAM-checking state, DNA-unwinding state, pre-catalytic state and catalytically active state, which illuminate notable differences in the activation mechanisms between small SaCas9 and larger SpCas9. Overall, our findings demonstrate that eSaCas9-NNG could be used as a versatile genome editing tool for in vivo gene therapy, and improve our mechanistic understanding of the diverse CRISPR-Cas9 nucleases.

Abstract The CRISPR/Cas12a system has revolutionized molecular diagnostics; however, its application in directly detecting complex structured RNA remains challenging. Recently, we have developed a RNA detection method called SCas12a, which exhibits high sensitivity and efficiency in detecting RNA molecules devoid of intricate secondary structures. Here, we present an enhanced SCas12a assay (SCas12aV2) that facilitates precise amplification-free detection of highly structured RNA molecules. Our approach reengineers the split Cas12a system by optimizing the scaffold RNA length and targeting asymmetric RNA structures, thereby minimizing steric hindrance. We observe that utilization of a dsDNA-ssDNA hybrid DNA activator significantly enhances both the sensitivity and kinetics compared to those achieved using traditional ssDNA or dsDNA activators. The SCas12aV2 assay demonstrates exceptional sensitivity, with a limit of detection reaching 246 aM for pooled activators and 10 pM for single-site targeting. It also exhibits high specificity for single nucleotide polymorphisms (SNPs) and successfully identifies viable bacterial populations and SARS-CoV-2 infections from clinical samples. The assay's versatility is further highlighted by its applicability to various Cas12a orthologs, including the thermostable CtCas12a. This work offers a significant advance in molecular diagnostics, enhancing the potential for accurate and efficient RNA detection.

The ATP-binding cassette (ABC) transporter superfamily, one of the largest membrane protein families, plays a crucial role in multidrug resistance (MDR) in cancer by mediating the efflux of various chemotherapeutic agents, thereby lowering their intracellular concentrations and diminishing therapeutic effectiveness. Beyond drug efflux, these transporters are also involved in vital biological processes, such as signal transduction in cancer. Over the past few decades, extensive structural and functional research has provided valuable insights into the broad substrate specificity and transport mechanisms of ABC transporters, leading to promising strategies for overcoming MDR. This review will provide a structural understanding of the interactions between ABC transporters and inhibitors to develop novel cancer therapeutics. Additionally, we focus on methods such as irradiation-based immune therapies, thermal therapies, nanomedicine, CRISPR-CAS, and natural therapies that can genetically modify ABC transporters to reduce their expression or reverse the drug efflux ability. Knowledge gained from these approaches can then be translated into the development of new cancer therapeutics that can combat chemotherapy resistance.

Summary Ustilago maydis is a biotrophic fungus infecting maize, secreting effector proteins to manipulate host cellular processes and create nutrient-rich environments for its growth. Three effectors, Hap1-3 (hypertrophy-associated proteins), were identified as virulence factors promoting hypertrophic mesophyll tumor cells (HTT). Immunoprecipitation and mass spectrometry revealed interactions among Hap effectors, suggesting potential effector complex formation. CRISPR-Cas9 triple knockout of hap1-3 demonstrated Hap1’s role in HTT formation. Infection assays identified Hap1 as a key virulence factor interacting with maize Snf1-related kinase 1 (SnRK1), a central energy regulator. RNA-seq analysis showed that Hap1 promotes cell cycle and starch biosynthesis genes, while CR- hap1 induced defense-related WRKY transcription factors. Phosphoproteomics revealed increased phosphorylation of SnRK1 and metabolic enzymes during SG200 infection. Our findings support a model where U. maydis induces hypertrophy through Hap1, targeting the SnRK1α subunit to prevent SnRK1 inhibition by high trehalose-6-phosphate (T6P), disrupting the antagonistic relationship between T6P and SnRK1. This reprograms host transcription to enhance starch metabolism and induce endoreduplication, leading to hypertrophy induction, while suppressing sugar-induced immune signaling.

One avenue to better understand brain evolution is to map molecular patterns of evolutionary changes in neuronal cell types across entire nervous systems of distantly related species. Generating whole-animal single-cell transcriptomes of three nematode species from the Caenorhabditis genus, we observed a remarkable stability of neuronal cell type identities over more than 45 million years of evolution. Conserved patterns of combinatorial expression of homeodomain transcription factors are among the best classifiers of homologous neuron classes. Unexpectedly, we discover an extensive divergence in neuronal signaling pathways. While identities of neurotransmitter-producing neurons (glutamate, acetylcholine, GABA and several monoamines) remain stable, ionotropic and metabotropic receptors for all these neurotransmitter systems show substantial divergence, resulting in more than half of all neuron classes changing their capacity to be receptive to specific neurotransmitters. Neuropeptidergic signaling is also remarkably divergent, both at the level of neuropeptide expression and receptor expression, yet the overall dense network topology of the wireless neuropeptidergic connectome remains stable. Novel neuronal signaling pathways are suggested by our discovery of small secreted proteins that show no obvious hallmarks of conventional neuropeptides, but show similar patterns of highly neuron-type-specific and highly evolvable expression profiles. In conclusion, by investigating the evolution of entire nervous systems at the resolution of single neuron classes, we uncover patterns that may reflect basic principles governing evolutionary novelty in neuronal circuits.

Background Recent studies highlight the critical role of microglia in neurodegenerative disorders, and emphasize the need for humanized models to accurately study microglial responses. Human-mouse microglia xenotransplantation models are a valuable platform for functional studies and for testing therapeutic approaches, yet currently those models are only available for academic research. This hampers their implementation for the development and testing of medication that targets human microglia. Methods We developed the hCSF1 Bdes mouse line suitable as new transplantation model available to be crossed to any disease model of interest. The hCSF1 Bdes model created by CRISPR gene editing is RAG2 deficient and expresses human CSF1. Additional we crossed this model with two humanized App KI mice, the App Hu and the App SAA . Flow cytometry, immunohistochemistry and bulk sequencing was used to study the response of microglia in the context of Alzheimer’s disease . Results Our results demonstrate the successful transplantation of iPSC-derived human microglia into the brains of hCSF1 Bdes mice without triggering a NK-driven immune response. Furthermore we confirmed the multipronged response of microglia in the context of Alzheimer’s disease. The hCSF1 Bdes and the crosses with the Alzheimer’s disease knock-in model App SAA and the humanized App knock-in control mice, App Hu are deposited with EMMA and fully accessible to the research community. Conclusion the hCSF1 Bdes mouse is available for both non-profit and for-profit organisations, facilitating the use of the xenotransplantation paradigm for human microglia to study complex human disease.

Defects in DNA single-strand break repair are associated with neurodevelopmental and neurodegenerative disorders. One such disorder is that resulting from mutations in XRCC1 , a scaffold protein that plays a central role in DNA single-strand base repair. XRCC1 is recruited at sites of single-strand breaks by PARP1, a protein that detects and is activated by such breaks and is negatively regulated by XRCC1 to prevent excessive PARP binding and activity. Loss of XRCC1 leads to the toxic accumulation and activity of PARP1 at single-strand breaks leading to base excision repair defects, a mechanism that may underlie pathological changes in patients carrying deleterious XRCC1 mutations. Here, we demonstrate that xrcc1 knockdown impairs development of the cerebellar plate in zebrafish. In contrast, parp1 knockdown alone does not significantly affect neural development, and instead rescues the cerebellar defects observed in xrcc1 mutant larvae. These findings support the notion that PARP1 inhibition may be a viable therapeutic candidate in neurological disorders.

Transposable elements (TEs) provide sequences that are powerful cis-regulatory drivers of gene expression programmes. This is particularly apparent during early development when many TEs become de-repressed. MERVL elements are highly yet transiently upregulated in mouse totipotent 2-cell (2C) embryos during major zygotic genome activation (ZGA), and in 2C-like cells in vitro. One of the most powerful activators of MERVL is the pioneer transcription factor, Dux. However, apparent differences lie in the requirement for Dux versus MERVL activation in embryos, for unclear reasons. Moreover, sustained Dux activation causes cell toxicity in multiple cell types, which may or may not be linked to MERVL activation. Using a CRISPR-activation, 2C-GFP reporter system, we have unpicked the relative role of Dux and MERVL in ZGA, totipotent-like characteristics and cell toxicity. We find that direct MERVL activation comprises only a portion of the Dux-dependent transcriptome, and which is sufficient for expanded fate potential, but not other totipotency features. Conversely, Dux-induced pathology is independent of MERVL activation and involves induction of the pro-apoptotic factor, Noxa. Our study highlights the complexity of the Dux-MERVL transcriptional network and uncovers a new player in Dux-driven pathology.

The extracellular matrix (ECM) is a mixture of glycoproteins and fibrous proteins that provide the biophysical properties necessary to maintain cellular homeostasis. ECM integrity is of particular importance during development, where it allows proper migration and cellular differentiation. Laminins are ECM heterotrimeric proteins consisting of α, β, and γ chains. There are five known α chains, four β chains, and three γ chains. Thus, there are 60 potential combinations for laminin trimers, however only 16 laminin trimers have been identified to date. Furthermore, none of them contain laminin β4 and its function is unknown. Here, we sought to characterize the role of LAMB4 (the gene encoding laminin β4) during human embryonic development of the peripheral sensory nervous system. Using human pluripotent stem cells (hPSCs), we found that LAMB4 is expressed in the ectoderm in the early stages of sensory neuron (SN) specification. SNs, part of the peripheral nervous system, are specialized neurons that detect pain, temperature, and touch. Surprisingly, more than 20 million people in the US have some form of peripheral nerve damage (including SNs), however there are very few treatment options available. Learning about the biology of peripheral neurons will uncover potential new therapeutic targets, thus we focused on understanding the effects of LAMB4 in SNs. First, we knocked out LAMB4 in hPSCs, using CRISPR/Cas9, and found that loss of LAMB4 impairs the migration of the SN progenitors neural crest cells (NCCs) and harms SN development and survival. To assess if LAMB4 has clinical relevance, we studied the genetic disorder Familial Dysautonomia (FD), which specifically affects the peripheral nervous system. FD is caused by a mutation in ELP1 (a component of the Elongator complex) leading to developmental and degenerative defects in SNs. A previous report showed that patients with severe FD harbor additional single nucleotide variants in LAMB4. We found that these variants sharply downregulate the expression of LAMB4 and laminin β4 levels in SNs differentiated from induced pluripotent stem cells (iPSCs) reprogramed from patients with severe FD. Moreover, a healthy ECM is sufficient to rescue the developmental phenotypes of FD, further confirming that ECM defects contribute significantly to the etiology of FD. Finally, we found that LAMB4/laminin β4 is necessary for actin filament accumulation and it interacts with laminin α4 and laminin γ3, forming the laminin-443, a previously unreported laminin trimer. Together, these results show that LAMB4 is a critical, but largely unknown gene required for SN development and survival.

Mapping enhancers and their target genes in specific cell types is crucial for understanding gene regulation and human disease genetics. However, accurately predicting enhancer-gene regulatory interactions from single-cell datasets has been challenging. Here, we introduce a new family of classification models, scE2G, to predict enhancer-gene regulation. These models use features from single-cell ATAC-seq or multiomic RNA and ATAC-seq data and are trained on a CRISPR perturbation dataset including >10,000 evaluated element-gene pairs. We benchmark scE2G models against CRISPR perturbations, fine-mapped eQTLs, and GWAS variant-gene associations and demonstrate state-of-the-art performance at prediction tasks across multiple cell types and categories of perturbations. We apply scE2G to build maps of enhancer-gene regulatory interactions in heterogeneous tissues and interpret noncoding variants associated with complex traits, nominating regulatory interactions linking INPP4B and IL15 to lymphocyte counts. The scE2G models will enable accurate mapping of enhancer-gene regulatory interactions across thousands of diverse human cell types.

Cupriavidus necator H16 is a promising microbial platform strain for CO 2 valorisation. While C. necator is amenable to genome editing, existing tools are often inefficient or rely on lengthy protocols, hindering its rapid transition to industrial applications. In this study, we simplified and accelerated the genome editing pipeline for C. necator by harnessing the Self-splicing Intron-Based Riboswitch (SIBR) system. We used SIBR to tightly control and delay Cas9-based counterselection, achieving >80% editing efficiency at two genomic loci within 48 hours after electroporation. To further increase the versatility of the genome editing toolbox, we upgraded SIBR to SIBR2.0 and used it to regulate the expression of Cas12a. SIBR2.0-Cas12a could mediate gene deletion in C. necator with ∼70% editing efficiency. Overall, we streamlined the genome editing pipeline for C. necator , facilitating its potential role in the transition to a bio-based economy.

48% of hereditary disease are caused by single C-to-T base conversion, which makes efficient A-to-G base editing tools (ABEs) have great potential in the treatment of these diseases. However, the existing efficient ABE, while catalyzing A-to-G conversion, will bring more A and C bystander editing and off-target events, which poses safety concerns for their clinical application. Here, we developed ABE10 (ABE8e with TadA-8e A48E) for efficient and accurate editing of As in YA motifs with YAY>YAR (Y=T or C, R=A or G) hierarchy through structure-oriented rational design. Compared with ABE3.1, currently the only motif (YAC) preference ABE version, ABE10 exhibited A-to-G editing efficiency improvement with an average up to 3.1-fold in indicated YA motif while maintaining reduced bystander Cs editing and minimized DNA or RNA off-targets. Also, we showed ABE10 corrected pathogenic mutation with high efficiency and precision in human cells. Moreover, by ABE10, we efficiently and precisely generated hypocholes-terolemia and tail-loss mouse models mimicking human associated disease and mouse PCSK9 base editing in vivo for hypercholesterolemia gene therapy, indicating their great potential in broad applications for and disease modeling and gene therapy.

Summary Our understanding of the factors underlying the evolutionary success of different lineages of pandemic Vibrio cholerae remains incomplete. Interestingly, two unique genetic signatures define the West African South American (WASA) lineage of V. cholerae responsible for the 1991-2001 Latin American cholera epidemic. Here we show these signatures encode diverse anti-phage defence systems. Firstly, the WASA-1 prophage encodes a 2-gene abortive-infection system WonAB that renders the lineage resistant to the major predatory vibriophage ICP1, which alongside other phages, is thought to restrict cholera epidemics and has potential for use in prophylaxis. Secondly, a unique set of genes on the Vibrio seventh pandemic island II encodes an unusual modification-dependent restriction system targeting phages with modified genomes, and a new member of the Shedu defence family that defends against vibriophage X29. Taken together, we propose that these anti-phage defence systems have likely contributed to the success of a major epidemic lineage of the ongoing seventh cholera pandemic.

Glioblastoma (GBM) is a highly aggressive primary malignant adult brain tumor that inevitably recurs with a fatal prognosis. This is due in part to metabolic reprogramming that allows tumors to evade treatment. We therefore must uncover the pathways mediating these adaptations to develop novel and effective treatments. We searched for genes that are essential in GBM cells as measured by a whole-genome pan-cancer CRISPR screen available from DepMap and identified the methionine metabolism genes MAT2A and AHCY . We conducted genetic knockdown, evaluated mitochondrial respiration, and performed targeted metabolomics to study the function of these genes in GBM. We demonstrate that MAT2A or AHCY knockdown induces oxidative stress, hinders cellular respiration, and reduces the survival of GBM cells. Furthermore, selective MAT2a or AHCY inhibition reduces GBM cell viability, impairs oxidative metabolism, and changes the metabolic profile of these cells towards oxidative stress and cell death. Mechanistically, MAT2a or AHCY regulates spare respiratory capacity, the redox buffer cystathionine, lipid and amino acid metabolism, and prevents DNA damage in GBM cells. Our results point to the methionine metabolic pathway as a novel vulnerability point in GBM. Significance We demonstrated that methionine metabolism maintains antioxidant production to facilitate pro-tumorigenic ROS signaling and GBM tumor cell survival. Importantly, targeting this pathway in GBM can potentially reduce tumor growth and improve survival in patients.

Genetic variations in MS4A4A and MS4A6A are linked to the regulation of cerebrospinal fluid soluble TREM2 (sTREM2) levels and are associated with Alzheimer’s disease (AD) risk and progression. Using CRISPR knockout and MS4A4A-degrading antibodies in primary human microglia, non-human primates (NHP), and a xenotransplantation model of amyloid pathology, we provide evidence that MS4A4A and MS4A6A are negative regulators of both the transmembrane and soluble TREM2 proteins. They also negatively regulate microglia proliferation, survival, metabolism, lysosomal function, energetics, phagocytosis, and disease-fighting states. Mechanistically, we find that MS4A4A exerts negative regulation by interacting with MS4A6A and protecting it from degradation. MS4A6A in turn forms a complex with and blocks the co-receptor DAP12, which is required for the stability, cell surface localization, and signaling of TREM2 and other receptors. Taken together, the data indicate that MS4A4A and MS4A6A are cooperating, post-transcriptional negative regulators of TREM2 and microglial function, and potential drug targets for AD.

Summary Background Environmental acquisition of Burkholderia pseudomallei can cause melioidosis, a life-threatening yet underreported disease. Understanding environmental exposure is essential for effective public health interventions, yet existing tools are limited in their ability to quantify exposure risks. Methods We conducted two complementary studies across a 15,118 km 2 area of northeast Thailand to improve detection methods and investigate risk factors for melioidosis. In the first study, we compared a newly developed, equipment-light CRISPR-based assay (CRISPR-BP34) with conventional culture methods using both spiked samples and real water samples from household and community sources (November 2020 - November 2021). The second study involved a case-control analysis of 1,135 participants (October 2019 - January 2023) to evaluate the association between environmental exposure to B. pseudomallei (detected in Study 1) and melioidosis risk. Findings The CRISPR-BP34 assay demonstrated improved sensitivity (93.52% vs 19.44% for conventional methods) and specificity (100% vs 97.98%), allowing for more accurate detection of B. pseudomallei and exposure risk quantification. Environmental exposure to B. pseudomallei in water sources within a 10 km radius of participants’ households was significantly associated with increased melioidosis risk (OR: 2.74 [95% CI 1.38-5.48]). This risk was also heightened by known factors: occupational exposure among agricultural workers (4.46 [2.91-6.91]), and health factors like elevated hemoglobin A1c, indicating diabetes (1.35 [1.19-1.31]). Interpretation Our findings underscore the impact of environmental contamination on melioidosis risk. The robust association between contaminated water sources, including piped water systems, and clinical cases highlights the urgent need for improved water sanitation to mitigate melioidosis risk. Funding Wellcome Trust Evidence before this study We conducted a PubMed search, without language restrictions from database inception to 11 September 2024, using the following search terms: (“ Burkholderia pseudomallei ” AND “environment* sampl*”) or (“ Burkholderia pseudomallei ” AND “spatial”), yielding 172 research and review articles. Several studies attempted to link the detection of B. pseudomallei in the environment with melioidosis risk through case-control and case-only designs. However, none demonstrated a statistically significant relationship between environmental presence of B. pseudomallei and infection risk (case-control) or clinical severity (case-only). The main challenges included low detection rates in environmental samples, inconsistent sampling methodologies, and outdated guidelines, which restricted the use of individual analyses or meta-analyses across combined studies. While soil is widely considered the natural reservoir for B. pseudomallei , its distribution varies significantly across soil textures, moisture levels, and depths, often leading to inconsistent or inconclusive data. These variations complicate efforts to establish a reliable link between soil contamination and melioidosis risk. Water sampling has been suggested as a viable alternative due to its more homogenous nature and simpler collection methods. Water also directly reflects human exposure risk, as people are regularly in contact with natural water bodies and treated water systems. However, detecting B. pseudomallei in water is challenging due to its low abundance. Molecular techniques such as PCR, following an enrichment process, have shown the highest sensitivity for detecting B. pseudomallei . The enrichment step enhances B. pseudomallei growth while suppressing competing microorganisms. For example, in a study conducted in a disease hotspot in Laos, positive detection rates improved from a median of 50% (IQR 42.5 - 53.8%) using conventional culture inspection methods, to 55% using PCR alone, and 75% with PCR following enrichment. While this approach is promising, it requires access to PCR equipment, which is often unavailable in resource-limited, melioidosis-endemic regions. These challenges create gaps in current detection methods and hinder the ability to accurately quantify environmental exposure risks and identify high-risk areas. Added value of this study Our study addressed these gaps by developing an equipment-light device capable of detecting B. pseudomallei in environmental samples after enrichment. This approach eliminates the need for complex PCR equipment while maintaining high sensitivity and specificity, comparable to qPCR. Our findings established a statistically significant link between environmental exposure to B. pseudomallei within a 10 km radius of households and 2.74-fold increased odds [95% CI: 1.38-5.48] of acquiring melioidosis. This risk remains significant even after adjusting for confounding factors such as underlying health conditions (e.g. diabetes) and occupational exposures. Implications of all the available evidence Our results confirm that B. pseudomallei can be detected in both natural water reservoirs and publicly treated piped water systems in endemic regions, and that its presence is positively associated with the occurrence of melioidosis. The detection of B. pseudomallei in treated water systems emphasises the urgent need for improved water sanitation measures. These results highlight the importance of environmental monitoring and targeted interventions to reduce melioidosis risks in the endemic areas.

Mpox has emerged as a critical public health challenge, creating an urgent need for rapid, reliable, and field-deployable diagnostic tools for outbreak settings. Here, we present Kairo-CONAN, a novel CRISPR-Cas3-based point-of-care (POC) diagnostic platform for Mpox, engineered for sustainability and portability. This system leverages a disposable hand warmer (Kairo) as a stable heat source and incorporates freeze-dried reagents for ambient temperature stability, enabling device-free, sensitive detection through lateral flow assay strips. Utilizing CRISPR-Cas3’s unique DNA-targeting and cleavage properties, we optimized probe DNA configurations for high specificity and designed clade-specific target crRNAs. Kairo-CONAN demonstrated rapid, high-sensitivity, and specific detection of Mpox virus (MPXV) DNA across multiple clades, including Clade Ia (Congo), Clade Ib (synthetic DNA), and Clade IIb (Tokyo). By addressing logistical and environmental challenges, Kairo-CONAN offers a sustainable, cost-effective, and field-adapted solution for infectious disease diagnostics, aligning with the 100-day mission framework to enhance global outbreak response efforts.

Early detection of HIV-1 infection is crucial to initiate anti-retroviral therapy (ART) to suppress viremia and disease progression. Herein, we developed a CRISPR/Cas12a-based HIV-1 detection assay by optimizing components for a coupled isothermal preamplification by recombinase polymerase amplification (RPA). The HIV-1 Indian Clade-C-specific conserved pol region was targeted by crRNA designed for Clade-specific detection. The CRISPR/Cas12a cleavage of the viral cDNA input is displayed as a single visually detectable outcome due to the collateral cleavage of the ssDNA-FAM-BQ reporter, enabling the rapid detection of HIV-1. The performance of the assay was evaluated by testing sera of 41 Indian Clade C HIV-1 seropositive individuals, which included 28 HIV-1 infected infant samples, HIV-1 Indian clade C genome plasmid, viral disease control DNA/RNA samples (Influenza, RSV, Parvovirus, HPIV, CMV, and HBV), and 31 healthy donor sera samples. With 96% sensitivity and 92.65% specificity for HIV-1C detection, with fluorescence and visual readout, and a capability of detection using lateral flow dipsticks, our CRISPR/Cas12a-based HIV-1 C detection assay demonstrates the potential to be developed into a robust point-of-care molecular diagnostic test for HIV-1C. Moreover, it may serve as a potential rapid NAT alternative in detecting mother-to-child transmission (MCT) of HIV-1C in infants (<2 years of age), where rapid antibody-based serology tests are rendered ineffective due to the presence of maternal antibodies.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), an exceptionally potent genome-editing technique developed in 2012, is the ideal tool of the future for treating diseases by permanently correcting deleterious base mutations or disrupting disease-causing genes with great precision and efficiency. However, it is prone to cleaving double-stranded DNA in off-target genes and generating random mutations in the process. These drawbacks restrict its application in fundamental research and agriculture, and raises safety concerns in the field of medicine. Fortunately, the new gene editing technology derived from CRISPR/Cas9, known as prime editing, has the potential to provide targeted sequence insertion, deletion, and transversion, all while avoiding the formation of double-strand breaks, thus minimizing adverse effects. Meanwhile, the rapid development of this technology makes its application wider and broader. This review summarizes the current developments and optimizations of the prime editing (PE) system with improved editing efficiency and precision. Along with discussing the most recent delivery techniques and outlining the PE applications that are being used both in vitro and in vivo.

CRISPR-Cas nucleases are transforming genome editing, RNA editing, and diagnostics but have been limited to RNA-guided systems. We present ΨDNA, a DNA-based guide for Cas12 enzymes, engineered for specific and efficient RNA targeting. ΨDNA mimics a crRNA but with a reverse orientation, enabling stable Cas12-RNA assembly and activating trans-cleavage without RNA components. ΨDNAs are effective in sensing short and long RNAs and demonstrated 100% accuracy for detecting HCV RNA in clinical samples. We discovered that ΨDNAs can guide certain Cas12 enzymes for RNA targeting in cells, enhancing mRNA degradation via ribosome stalling and enabling multiplex knockdown of multiple RNA transcripts. This study establishes ΨDNA as a robust alternative to RNA guides, augmenting the potential of CRISPR-Cas12 for diagnostic applications and targeted RNA modulation in cellular environments.