Summary Mitochondria contain their own genome, the mitochondrial DNA (mtDNA), which is under strict control of the cell nucleus. mtDNA occurs in many copies in each cell, and mutations often only affect a proportion of them, giving rise to heteroplasmy. mtDNA copy number and heteroplasmy level together shape the cell- and tissue-specific impact of mtDNA mutations, ultimately giving rise to rare mitochondrial and common neurodegenerative diseases. However, little is known about how copy number and heteroplasmy interact within single cells, and how this is regulated by the nuclear genes and pathways that sense and control them. Here we describe MitoPerturb-Seq for CRISPR/Cas9-based high-throughput single-cell interrogation of the impact of nuclear gene perturbation on mtDNA copy number and heteroplasmy. We screened a panel of nuclear mtDNA maintenance genes in cells with heteroplasmic mtDNA mutations. This revealed both common and perturbation-specific aspects of the integrated stress-response to mtDNA depletion, that were only partially mediated by Atf4, and caused cell-cycle stage-independent slowing of cell proliferation. MitoPerturb-Seq thus provides novel experimental insight into disease-relevant mito-nuclear interactions, ultimately informing development of novel therapies targeting cell- and tissue-specific vulnerabilities to mitochondrial dysfunction.

The transcription factor (TF) GATA4 is a key mediator of cardiogenesis. GATA4 regulates cardiogenesis through the expression of its target genes, only some of which have been identified. We have used a gain of function model based on pluripotent embryonic ectoderm explants from Xenopus embryos expressing GATA4, to identify a set of downstream targets of GATA4 which are also regulated by Nodal, a known cardiogenic signal. GATA4 was shown to be required for the expression of target genes tbx2 and prdm1 in vivo, likely acting in a direct fashion by interacting with their regulatory regions. In addition, tbx2 and prdm1 are shown to have roles of their own in vivo, as downregulation of tbx2 , a positive target, and overexpression of prdm1 , a negative target, interferes with cardiac development in Xenopus embryos. The conservation of the GATA4-TBX2-PRDM1 regulatory relationship was shown in human iPSC-derived cardiomyocytes. Loss of function of GATA4 lead to downregulation of TBX2 , upregulation of PRDM1 expression and failure of cardiogenesis. GATA4-deficient cells failed to form normal cardiomyocytes, with most cells adopting alternative fates and only a small minority expressing an aberrant cardiomyocyte phenotype. Genome-wide transcriptomic analysis documented severe reduction of cardiomyocyte and endothelial cell transcriptomes and upregulation of transcriptional profiles of smooth muscle cells and fibroblasts. Disruption of TBX2 function did not alter cardiomyocyte differentiation efficiency but led to the formation of hypertrophic cardiomyocytes characterised by defective sarcomeres and deficient calcium signalling. In addition, we show that whilst PRDM1 is not essential for formation of cardiomyocytes it is implicated in suppression of alternative cell fates. The results presented establish a conserved regulatory relationship between GATA4 and its target genes TBX2 and PRDM1 and roles for these genes in the modulation of cardiomyocyte development, expanding the cardiac gene regulatory network and providing further insight into how cardiogenesis proceeds.

The MAST family of serine/threonine kinases has been implicated in a spectrum of human neurodevelopmental disorders. However, little is known about their biological function or regulation. Seeking to fill these gaps in our knowledge, we have identified upstream and downstream partners of MAST1. 14-3-3η, a neuronal 14-3-3 paralog, specifically interacts with MAST1 at two regulatory serines, S90 and S161. PAK, a neuronal regulator of the actin cytoskeleton, phosphorylates MAST1 to regulate its interaction with 14-3-3η. Exploiting mouse models of human Mega Corpus Callosum Syndrome (MCC) and whole brain phosphoproteomics, we identify the microtubule-associated protein Tau as a substrate of MAST1. We show that pathogenic MAST1 mutations perturb protein function either through misfolding or attenuation of kinase activity. Our data is consistent with a model in which the MAST kinases couple PAK, a neuronal regulator of the actin cytoskeleton, to microtubule remodeling during the differentiation and specification of cortical neurons.

Harnessing the precision of CRISPR systems for diagnostics has transformed nucleic acid detection. However, the integration of upstream cellular signals into CRISPR-based circuits remains largely underexplored. Here, we introduce a synthetic transduction platform that directly links endogenous DNA repair activity to CRISPR-Cas12a activation. By coupling base excision repair (BER) events to a programmable DNA-based transducer, our system converts the activity of DNA glycosylases, such as uracil-DNA glycosylase (UDG) and human 8-oxoguanine glycosylase (hOGG1), into a robust fluorescence signal via Cas12a-mediated trans-cleavage. This one-step CRISPR-based assay operates directly in cell lysates, enabling rapid and sensitive readout of enzymatic activity with high specificity. Additionally, it also enables in 15 minutes the throughput screening of novel potential inhibitors with high sensitivity. The modular design allows adaptation to diverse repair enzymes, offering a generalizable strategy for transforming intracellular repair events into programmable outputs. This approach lays the foundation for activity-based molecular diagnostics, synthetic gene circuits responsive to cellular states, and new tools for monitoring DNA repair in real time and drug screening.

Noncoding genetic variants underlie many complex diseases, yet identifying and interpreting their functional impacts remains challenging. Late-onset Alzheimer’s disease (LOAD), a polygenic neurodegenerative disorder, exemplifies this challenge. The disease is strongly associated with noncoding variation, including common variants enriched in microglial enhancers and rare variants that are hypothesized to influence neurodevelopment and synaptic plasticity. These variants often perturb regulatory sequences by disrupting transcription factor (TF) motifs or altering local TF interactions, thereby reshaping gene expression and chromatin accessibility. However, assessing their impact is complicated by the context-dependent functions of regulatory sequences, underscoring the need to systematically examine variant effects across diverse tissues, cell types, and cellular states. Here, we combined in vitro and in vivo massively parallel reporter assays (MPRAs) with interpretable machine-learning models to systematically characterize common and rare variants across myeloid and neural contexts. Parallel profiling of variants in four immune states in vitro and three mouse brain regions in vivo revealed that individual variants can differentially and even oppositely modulate regulatory function depending on cell-type and cell-state contexts. Common variants associated with LOAD tended to exert stronger effects in immune contexts, whereas rare variants showed more pronounced impacts in brain contexts. Interpretable sequence-to-function deep-learning models elucidated how genetic variation leads to cell-type-specific differences in regulatory activity, pinpointing both direct transcription-factor motif disruptions and subtler tuning of motif context. To probe the broader functional consequences of a locus prioritized by our reporter assays and models, we used CRISPR interference to silence an enhancer within the SEC63-OSTM1 locus that harbors four functional rare variants, revealing its gatekeeper role in inflammation and amyloidogenesis. These findings underscore the context-dependent nature of noncoding variant effects in LOAD and provide a generalizable framework for the mechanistic interpretation of risk alleles in complex diseases.

Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. We evaluated CasX2 ( Plm Cas12e), a class II CRISPR system derived from Planctomycetes , a non-pathogenic bacterium present in aquatic and terrestrial soils. CasX2 offers several advantages over Streptococcus pyogenes Cas9 ( Sp Cas9) and Staphylococcus aureus Cas9 ( Sa Cas9), including its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and no known pre-existing immunity in humans. A recent study reported that a three amino acid substitution in CasX2 significantly enhanced cleavage activity (1). Therefore, we compared cleavage efficiency and double-stranded break repair characteristics between the native CasX2 and the variant, CasX2 Max , for cleavage of CCR5 , a gene that encodes the CCR5 receptor important for HIV-1 infection. Two CasX2 single guide RNAs (sgRNAs) were designed that flanked the 32 bases deleted in the natural CCR5 Δ32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2 Max using sgRNAs with 20 nt and 23 nt spacer lengths, enabled robust genomic cleavage of CCR5 . Structural modeling indicated that two of the CasX2 Max substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2 Max in cellular gene excision. In sum, CasX2 Max consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.

The rapid advancement of genetic editing technologies, such as CRISPR-Cas9, has introduced unprecedented opportunities and challenges within professional sports. This study aims to systematically evaluate the legal and ethical implications associated with the application of gene editing among elite athletes. Employing a mixed-methods design, we conducted a comprehensive survey of 312 stakeholders-including athletes, coaches, legal experts, and ethicists-across five continents. Advanced statistical analyses, including Structural Equation Modeling (SEM) and Multivariate Logistic Regression, were utilized to identify significant predictors of legal risk perception and ethical concern. Results reveal a pronounced divergence in stakeholder attitudes: while 68% of legal professionals emphasize regulatory gaps, 74% of athletes express uncertainty regarding long-term health consequences. The SEM model demonstrated that perceived fairness (β=0.41, p<0.001) and regulatory clarity (β=0.36, p<0.001) are the strongest predictors of overall acceptance. These findings underscore the urgent need for robust international frameworks to address the multifaceted risks of gene editing in sports and highlight the importance of transparent policy-making. Our research provides actionable insights for regulators, sports organizations, and bioethics committees to anticipate and manage the evolving landscape of genetic technologies in athletics.

Leishmania amastigotes ingested by female phlebotomine sand flies are exposed to a harsh and dynamic environment that differs markedly from the intracellular niche in the mammalian host in temperature, pH and nutrient availability. Membrane transporter proteins, channels and pumps play a crucial role in maintaining cellular physiology under changing environments. A systematic loss-of-function screen of the L. mexicana transporter deletion mutants in macrophage and mouse infections previously identified transporter genes important for the amastigote stage. To test which transporters are important for the promastigote stage in the insect vector, we measured the fitness of gene deletion mutants in Lu. longipalpis sand flies. Pooled libraries of different complexities, consisting of 71 to 317 barcoded parasite lines allowed for an estimation of the bottleneck size in experimental infections, providing a foundation for similar experimental bar-seq studies. The fitness of each mutant parasite line was measured by tracking population composition over a course of 9 days in the sand flies and compared with the growth fitness of promastigotes over 7 days in laboratory cultures. There was a high correlation of fitness scores in vitro and in vivo , but 34 mutants showed a loss of fitness only in vivo , including deletion mutants of vacuolar H+ ATPase (V-ATPase) subunits. V-ATPase deletion mutants expressed low levels of the metacyclic-specific transcript sherp in vitro and failed to generate metacyclic promastigotes in sand flies, indicating that V-ATPase function is required for parasite differentiation and progression through the Leishmania life cycle. Author Summary Leishmania parasites cause leishmaniases - a group of neglected tropical diseases that affect millions of people worldwide. These parasites must survive in two radically different environments: inside a mammalian host and within the gut of a blood-feeding sand fly. To thrive in the sand fly, Leishmania undergo extensive physiological changes and depend on transporter proteins to move nutrients and other molecules across their cell membranes. In this study, we focused on identifying which of these transporters are critical for the parasite’s survival inside the sand fly. We used a library of genetically engineered Leishmania promastigotes - the parasite form adapted to the insect vector - to assess the importance of more than 300 different transporter genes. We discovered that 34 of these transporters are essential for successful colonization of the sand fly. Among them, one key protein complex - the vacuolar H + ATPase (V-ATPase) pump – was found to be crucial for parasite survival in the insect vector. Our findings deepen our understanding of how Leishmania adapts to life within the sand fly and highlight potential molecular targets for disrupting its transmission.

Lung cancer histological subtypes include lung adenocarcinoma (LUAD) and small cell lung cancer (SCLC). While typically distinct, combined LUAD/SCLC histology tumors occur, and LUAD can transform into SCLC as a resistance mechanism to targeted therapies, especially in EGFR -Mutant LUADs with RB1 / TP53 -inactivation. Although PRC2 complex expression increases during this transformation, its functional role has remained unclear. Using CRISPR-based autochthonous immunocompetent GEMMs, we demonstrate that inactivation of EED, the core PRC2 scaffolding subunit, impairs SCLC tumorigenesis and drives histological transformation from ASCL1-positive SCLC to LUAD through a transient NEUROD1-positive intermediate state. Mechanistically, EED loss de-represses bivalent genes co-marked by H3K27me3 and H3K4me3, including LUAD oncogenic RAS, PI3K, and MAPK pathway genes, to promote transformation to LUAD. Consistently, these same signaling genes are bivalently repressed in human SCLC patient-derived xenograft (PDX) tumors, suggesting a conserved PRC2-dependent mechanism to repress LUAD lineage oncogenic signaling to maintain the SCLC neuroendocrine identity. In a complementary EGFR -mutant LUAD GEMM with RB1/TP53 inactivation, EED was required for LUAD-to-SCLC transformation and distant metastasis upon EGFR withdrawal. These findings identify the PRC2 complex as a key epigenetic enforcer of SCLC neuroendocrine identity and nominate EED inhibition as a potential strategy to block SCLC transformation in high-risk LUAD.

Bacteria encode an enormous diversity of defense systems including restriction-modification and CRISPR-Cas that cleave nucleic acid to protect against phage infection. Bioinformatic analyses demonstrate many recently identified anti-phage defense operons are comprised of a predicted nuclease and an accessory NTPase protein, suggesting additional classes of nucleic acid targeting systems remain to be understood. Here we develop large-scale comparative cell biology and biochemical approaches to analyze 16 nuclease-NTPase systems and define shared features that control anti-phage defense. Purification, biochemical characterization, and in vitro reconstitution of nucleic acid targeting for each system demonstrate protein–protein complex formation is a universal feature of nuclease-NTPase systems and explain patterns of phage targeting and susceptibility. We show that some nuclease-NTPase systems use highly degenerate recognition site preferences to enable exceptionally broad nucleic acid degradation. Our results uncover shared principles of anti-phage defense system function and provide a foundation to explain the widespread role of nuclease-NTPase systems in bacterial immunity.

Abstract Protein-glutamine glutaminases (PGs; EC 3.5.1.44) have gained attention in the food industry due to their application in plant protein products. The recently discovered PG from Bacteroides helcogenes (PGB) has especially been shown to provide promising characteristics for improving the techno-functional properties of plant proteins. A prerequisite for food enzymes, such as the PG, is their production with an expression host that meets food safety and yield requirements. The antibiotic-free and secretory production of the PGB was targeted in this study using the isolated Bacillus subtilis 007. The CRISPR/Cas9-mediated approach enabled specific genomic PGB integrations, while simultaneously deleting unwanted B. subtilis traits. Firstly, the PGB expression cassette was integrated into the sigF gene, leading to an asporogenic strain and extracellular activity of 4.1 µkat/L culture in bioreactor cultivations. However, excessive foaming hampered the production process tremendously. Consequently, a second PGB copy was integrated into the sfp locus, which is involved in the production of lipopeptides, such as surfactin. As a result, the PGB activity was increased to 5.4 µkat/L culture, and foaming during cultivation was reduced significantly. The introduction of a third PGB copy for preventing cell motility did not increase production, however, the integration into the well-established amyE locus improved the PGB yield during reactor cultivations. A final extracellular activity of 9.5 µkat/L culture was reached. The multiple genomic integrations of the PGB gene enabled the efficient PGB secretion in an optimized B. subtilis host without the need for antibiotics.

Drosophila immunity has been the focus of intense study and has impacted other research fields including innate immunity and agriculturally or epidemiologically relevant investigations of insect pests and vectors. Unsurprisingly for such a large body of work, some published results were later found to be irreproducible. Although some results have been contradicted in the literature, many have no published follow-up, either due to a lack of research or low motivation to publish negative or contradictory results. We have addressed this by performing a reproducibility project that analyses the verifiability of claims from articles published on Drosophila immunity before 2011. To assess reproducibility, we extracted claims from 400 articles on the Drosophila immune response to bacteria and fungi and performed preliminary verification by comparing these claims to other published literature in the field. Using alternative approaches, we also experimentally tested some ‘unchallenged’ claims, which had no published follow-up. The intent of this analysis was to centralize evidence on insights and findings to improve clarity for scientists that may base research programs on these data. All our data are published on a publicly available website associated with this article ( https://ReproSci.epfl.ch/ ) that encourages community participation. This article provides a short summary of claims that were found to have contradictory evidence, which may help the community to assess past findings on Drosophila immunity and improve clarity going forward.

Therapeutic modalities to programmably increase protein production are in critical need to address diseases caused by deficient gene expression via haploinsufficiency. Restoring physiological protein levels by increasing translation of their cognate mRNA would be an advantageous approach to correct gene expression, but has not been evaluated in an in vivo disease model. Here, we investigated if a translational activator could improve phenotype in a Dravet syndrome mouse model, a severe developmental and epileptic encephalopathy caused by SCN1a haploinsufficiency, by increasing translation of the SCN1a mRNA. We identifiy and engineere human proteins capable of increasing mRNA translation using the CRISPR-Cas Inspired RNA-targeting System (CIRTS) platform to enable programmable, guide RNA (gRNA)-directed translational activation with entirely engineered human proteins. We identify a compact (601 amino acid) CIRTS translational activator (CIRTS-4GT3), that can drive targeted, sustained translation increases up to 100% from three endogenous transcripts relevant to epilepsy and neurodevelopmental disorders. AAV-delivery of CIRTS-4GT3 targeting SCN1a mRNA to a Dravet syndrome mouse model led to increased SCN1a translation and improved survivability and seizure threshold - key phenotypic indicators of Dravet syndrome. This work validates a new strategy to address SCN1a haploinsufficiency and emphasizes the preclinical potential translational activation has to address neurological haploinsufficiency.