The increasing global prevalence of Mycobacterium abscessus infections presents a significant clinical challenge due to the pathogen’s intrinsic resistance to multiple antibiotics and poor treatment outcomes. Despite the necessity of genetic tools for studying its physiology, pathogenesis, and drug resistance, efficient methods for large-fragment deletions remain underdeveloped. Here, we report a CRISPR/Cas9-based dual-sgRNA system employing Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9), enabling efficient large-fragment knockout in M. abscessus with deletion efficiencies exceeding 90% at certain loci and spanning up to 16.7 kb. Furthermore, we systematically optimized the modular arrangement of genetic components in Cas9/dual-sgRNA expression plasmids and refined their construction workflow, achieving a significant reduction in cassette loss rates while enabling single-step plasmid assembly. Notably, deletion efficiency was position-dependent rather than correlated with target size, suggesting an influence of chromatin structure on editing outcomes. As the first CRISPR/Cas9-based platform capable of kilobase-scale deletions in M. abscessus , this system advances functional genomics studies and facilitates targeted investigations into virulence and antibiotic resistance mechanisms.

CRISPR/Cas12a-based assays, when integrated with lateral flow tests (LFTs), provide highly specific nucleic acid detection in a simple, rapid, and equipment-free format. Nevertheless, traditional DNA probes utilized for cleavage by Cas12a have notable limitations as the cleaved probe only has one label. To overcome this challenge, we engineered a novel type of DNA probe with multiple fluorescein (FAM) labels and a biotin-labeled single-stranded DNA fragment (polyFAM probe). The cleaved polyFAM parts of probes were detected using a specially designed sandwich LFT, where FAM-specific antibodies were immobilized in the test zone and conjugated with gold nanoparticles. The LFT ensured accurate recognition of the cleaved polyFAM fragments within 10 minutes. A comparison of five distinct polyFAM probes revealed that the highest signal-to-noise ratio was achieved with a tripod-branched probe synthesized via trebler phosphoramidite modification. Each arm of the tripod probe consists of a hexaethylene glycol spacer ending in a FAM label. Upon Cas12a cleavage, the tripod structure carrying three FAMs is released and detected by LFT. A rapid magnetic separation strategy was subsequently implemented, facilitating the efficient removal of uncleaved probes via biotin–streptavidin capture within 5 minutes. The CRISPR/Cas12a–tripod–LFT strategy demonstrated excellent sensitivity without preamplification, with a detection limit of 1.4 pM for DNA target of Salmonella Typhimurium. The CRISPR/Cas12a-tripod-LFT with preliminary loop-mediated isothermal amplification enabled the detection of as few as 0.3 cells per reaction. This innovative tripod probe with corresponding LFT creates a universal, sensitive, rapid, and equipment-free biosensing platform for CRISPR/Cas12a-based diagnostics in point-of-care applications.

Host cells contest invasion by intracellular bacterial pathogens with multiple strategies that recognise and / or damage the bacterial surface. To identify novel host defence factors targeted to intracellular bacteria, we developed a versatile proximity biotinylation approach coupled to quantitative mass spectrometry that maps the host-bacterial interface during infection. Using this method, we discovered that intracellular Shigella and Salmonella become targeted by UFM1-protein ligase 1 (UFL1), an E3 ligase that catalyses the covalent attachment of Ubiquitin-fold modifier 1 (UFM1) to target substrates in a process called UFMylation. We show that Shigella antagonises UFMylation in a dual manner: first, using its lipopolysaccharide (LPS) to shield from UFL1 recruitment; second, preventing UFM1 decoration by the bacterial effector IpaH9.8. Absence of UFMylation leads to an increase of bacterial burden in both human cells and zebrafish larvae, suggesting that UFMylation is a highly conserved antibacterial pathway. Contrary to canonical ubiquitylation, the protective role of UFMylation is independent of autophagy. Altogether, our proximity mapping of the host-bacterial interface identifies UFMylation as an ancient antibacterial pathway and holds great promise to reveal other cell-autonomous immunity mechanisms.

ABSTRACT The fungal pathogen Candida albicans colonises the human gut where short-chain fatty acids (SCFAs) offer sources of carbon. This fungus harbours one of the largest microbial families of ATO (Acetate Transport Ortholog) genes, which encode putative SCFA transport proteins. Here, we generate C. albicans null mutants lacking individual or all known putative SCFA transporter genes and compare their phenotypes in vitro and in vivo . We show that blocking ATO function in C. albicans impairs SCFA uptake and growth, particularly on acetate. The uptake of acetate is largely dependent on a functional Ato1 (also known as Frp3/Ato3) and it is effectively abolished upon deletion of all ATO genes. We further demonstrate that deletion of the entire ATO gene family, but not inactivation of ATO1 alone, compromises the stable colonisation of C. albicans in the murine gastrointestinal tract following bacterial disruption by broad-spectrum antibiotics. Our data suggest that the ATO gene family has expanded and diversified during the evolution of C. albicans to promote the fitness of this fungal commensal during gut colonisation, in part through SCFA utilisation. IMPORTANCE The human gut is rich in microbial fermentation products such as SCFAs, which serve as key nutrients for both bacteria and fungi. C. albicans , a common fungal resident of the gut and a cause of opportunistic infections, carries an unusually large family of ATO genes. This study reveals that this ATO gene family is required for the efficient uptake of acetate, the most abundant SCFA in the gut, and for stable colonisation of the gut. These findings uncover a new layer of metabolic adaptation in fungal commensals of humans and suggest that transporter gene expansion can shape microbial fitness in response to environmental nutrient signals.

Abstract Aim ; The study was conducted to establish the association between CRISPR-Cas system in Pseudomonas aeruginosa clinical and environmental strains to the sensitivity to phages and antibiotics. Method; In this study, the occurrence and distribution of CRISPR-Cas system was analyzed across the 50 Pseudomonas aeruginosa strains. Further, to evaluate the role of CRISPR-Cas system as inhibitor of HGT, the role of CRISPR-Cas system in relation to susceptibility of Pseudomonas aeruginosa to various antibiotics and phages was studied. Result : A total of 5 different types of Type I CRISPR- Cas systems i.e. Type IF, Type IE, Type IC, Type IV and Type IU were identified in P. aeruginosa strains. A total of 5 (17%) clinical strains and 8 (40%) environmental strains were positive for both CRISPR and cas3 gene. The analysis of phage as well as antibiotic susceptibility of both clinical and environmental strains revealed 36% clinical strains and75% environmental strains were found to be resistant to phages. Antibiotic susceptibility results were in contrast to phage susceptibility as 50% clinical strains were resistant to at least 3 antibiotics i.e. they were multidrug resistant (MDR) while only 15% environmental strains were classified as MDR. Conclusion : The phage susceptibility and antibiotic resistance data was found to be correlating to presence of CRISPR-Ca system in the P. aeruginosa .

Resistance to androgen receptor (AR)-targeted therapies, such as enzalutamide, in castration-resistant prostate cancer (CRPC) remains a significant clinical challenge, often driven by mechanisms including lineage plasticity. The precise molecular mechanisms driving this process, particularly downstream effectors, remain incompletely understood. Given its established roles in cell fate and stemness, alongside its complex functions in prostate cancer, the Notch signaling pathway presented a compelling focus for study. This study investigates the role of Notch signaling in mediating lineage plasticity and therapeutic resistance in CRPC. Employing transcriptomic analysis and functional assays, we identified Notch activity is elevated across prostate cancer progression resistance. Notably, both CRISPR-mediated knockout and targeted inhibition of Notch reversed enzalutamide resistance in vitro . Collectively, this study delineates dynamic alterations in Notch signaling activity during prostate cancer progression and establishes its function as a crucial and druggable driver of therapy resistance. These findings underscore Notch signaling as a promising therapeutic target to counteract resistance to AR-targeted therapies in advanced prostate cancer.

Transmission of Plasmodium parasites to the Anopheles vector critically depends on swift activation of mature gametocytes upon entry into the mosquito midgut. Induction of gametogenesis requires two simultaneous stimuli, a temperature drop and xanthurenic acid. Previous work in the murine malaria model Plasmodium yoelii identified a protein, termed gametogenesis essential protein ( GEP1 ), with a suggested role in xanthurenic acid-dependent activation of gametes. Here, we present an experimental genetics characterization of GEP1 in the human pathogen Plasmodium falciparum . Using CRISPR-Cas9 gene editing we generated PfGEP1 loss-of-function lines and analyzed their progression until gametocyte maturation. We show a complete defect in both male and female gametogenesis caused by disruption of PfGEP1 . Pfgep1(-) gametocytes do not produce gametes when activated with xan-thurenic acid or a drop in temperature. This defect could not be overcome by the phosphodiesterase inhibitor Zaprinast, which induces gametogenesis. We also explored GEP1 haplotypes in P. falciparum parasites circulating in endemic regions and show the presence of two non-synonymous SNPs, resulting in V241L and S263P mutations, in 12% and 20% of 49 sentinel samples, respectively. Together, our data indicate that GEP1 plays a central role in the gamete activation process independent of xanthurenic acid and validates Pf GEP1 as a promising transmission blocking target.

We present a generalisable, interpretable machine learning framework for therapeutic target discovery using single-cell transcriptomics, protein interaction networks, and drug proximity analysis. The pipeline integrates feature selection via gradient boosting classifiers, systems-level network inference, and in silico drug repurposing, enabling the identification of actionable targets with cellular specificity. As a proof of concept, we apply the method to clear cell renal cell carcinoma (ccRCC), an aggressive kidney cancer with limited treatment options. The model identifies 96 tumour-intrinsic genes, refines them to 16 targets through CRISPR screens and biological curation, and prioritises FDA-approved compounds via network-based proximity scoring. Several novel therapeutic mechanisms - including ABL1, CDK4/6, and JAK inhibition - emerge from this analysis, with predicted compounds showing superior efficacy to standard-of-care drugs across multiple ccRCC cell lines. Beyond ccRCC, this framework offers a scalable strategy for drug discovery across diverse diseases, combining machine learning interpretability with systems biology to accelerate therapeutic development.

Advances in genome engineering and single-cell RNA sequencing (scRNAseq) have revolutionized the ability to precisely map gene functions, yet scaling these techniques for large-scale genetic screens in animals remains challenging. We combined high-throughput gene disruption in zebrafish embryos via Multiplexed Intermixed CRISPR Droplets with phenotyping by multiplexed scRNAseq (MIC-Drop-seq). In one MIC-Drop-seq experiment, we intermixed and injected droplets targeting 50 transcriptional regulators into 1,000 zebrafish embryos, followed by pooled scRNAseq. Tissue-specific gene expression and cell abundance analysis of demultiplexed mutant cells recapitulated many known phenotypes, while also uncovering novel functions in brain and mesoderm development. We observed pervasive cell-extrinsic effects among these phenotypes, highlighting how whole-embryo sequencing captures complex developmental interactions. Thus, MIC-Drop-seq provides a powerful and scalable platform for mapping gene functions in vertebrate development with cellular resolution.

The penetrant PRKCA D463H mutation, a biomarker and potential driver in chordoid glioma, was found to provoke the development of chondrosarcomas in heterozygous knock-in mice. This mutation entirely abrogates kinase activity, but strikingly no oncogenic phenotype is observed for the related inactivating mutation D463N indicating that the lack of activity is not the driver. In cells, the D463H mutant closely mirrored PKCα WT behaviours and retained ATP binding, contrary to the related D463N mutant. Mechanistically, the PKCα D463H mutant protein was found to display quantitative alterations to the PKCα interactome, enhancing association with epigenetic regulators. This aligned with transcriptomic changes which resembled an augmented PKCα expression program, with enhanced BRD4, Myc and TGFβ signatures. D463H dependent reduced sensitivity to the BET inhibitors JQ1 and AZD5153 indicates the functional importance of these pathways. The data show that D463H is a dominant gain-of-function oncogenic mutant, operating through a non-catalytic allosteric mechanism. One Sentence Summary A PKCα catalytic inactivating mutation confers gain-of-function properties - a paradigm shift in kinase actions.

Abstract Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor in the gastrointestinal tract. In recent years, secondary resistance to the first-line drug imatinib has become its bottleneck of targeted therapy due to the unclear mechanism. It has important clinical significance for breaking through the bottleneck by screening and identifying the critical gene of imatinib resistance. Unbiased in vivo genome-wide genetic screening is a powerful approach to elucidate new molecular mechanisms. Here the genome-scale CRISPR/Cas9 Knockout Screening was applied to investigate imatinib resistance genes in GIST 882 cell line for two rounds, and it was found that deficiency of sphingosine 1-phosphate lyase coding gene SGPL1 can inhibit tumor cell apoptosis and accelerate cell cycle G1/S, finally leading to imatinib resistance in vitro and in vivo, by regulating the expression of Bcl-2, p27kip1 and p15INK4B via PI 3 K-Akt signaling pathway. In additionally, non-synonymous mutation in the exon of SGPL1 gene has been found by comparing the TCGA clinical drug resistance patient database. It was revealed that SPGL1 gene may be the critical gene of imatinib resistance. Taken together, our study provides a resource for achieving a deep understanding of the molecular basis of imatinib resistance.

Abstract CRISPR/Cas9 technology is an efficient tool for livestock gene editing. However, host genome function can be disrupted by the random integration of exogenous genes. To circumvent this issue, site-specific integration is required. This study established a multi-dimensional assessment system to evaluate the biological applicability of H11/Rosa26 safe harbor loci as targeted integration platforms for exogenous genes in goats. Donor cells carrying the enhanced green fluorescent protein (EGFP) reporter gene at the H11 and Rosa26 loci were generated via CRISPR/Cas9-mediated homology-directed repair; this was followed by somatic cell nuclear transfer to produce transgenic cloned embryos and healthy offspring. Multi-dimensional analyses revealed the following. At the cellular level, there was stable and efficient EGFP expression at integration sites, with donor cells maintaining normal cell cycle progression, proliferation capacity, and apoptosis levels, and with no alterations in the transcriptional integrity of adjacent genes. At the embryonic level, there was sustained EGFP expression across pre-implantation embryonic stages, with developmental metrics statistically indistinguishable from wild-type embryos. Finally, at the individual level, cloned offspring exhibited growth phenotypes consistent with wild-type counterparts, and EGFP showed broad-spectrum expression in eight tissues. This study provides the first demonstration of H11/Rosa26 loci as dual-functional sites that enable both high-efficiency integration and biosafety in goat models using a cross-scale (cellular-embryonic-individual) validation system, offering a precise and low-risk technical paradigm for livestock genetic improvement.

ABSTRACT Prokaryotes can acquire antivirus immunity via two fundamentally distinct types of processes: direct interaction with the virus as in CRISPR-Cas adaptive immunity systems and horizontal gene transfer (HGT) which is the main route of transmission of innate immunity systems. These routes of defense evolution are not mutually exclusive and can operate simultaneously, but empirical observations suggest that at least in some bacterial and archaeal species, one or the other route dominates the defense landscape. We hypothesized that the observed dichotomy stems from different life-history tradeoffs characteristic of these organisms. To test this hypothesis, we analyzed a mathematical model of a well-mixed prokaryote population under a stochastically changing viral prevalence. Optimization of the long-term population growth rate reveals two contrasting modes of defense evolution. In stable, predictable and fluctuating, unpredictable environments with a moderate viral prevalence, direct interaction with the virus and horizontal transfer of defense genes become the optimal routes of immunity acquisition, respectively. In the HGT-dominant mode, we observed a universal distribution of the fraction of microbes with different immune repertoires. Under very low virus prevalence, the cost of immunity exceeds the benefits such that the optimal state of a prokaryote is complete defense systems. By contrast, under very high virus prevalence, horizontal spread of defense systems dominates regardless of the stability of the virome. These findings might explain consistent but enigmatic patterns in the spread of antivirus defense systems among prokaryotes such as the ubiquity of adaptive immunity in hyperthermophiles contrasting their patchy distribution among mesophiles. IMPORTANCE The virus-host arms race is a major component of the evolutionary process in all organisms that drove the evolution of a broad variety of immune mechanisms. In the last few years, over 200 distinct antivirus defense systems have been discovered in prokaryotes. There are two major modes of immunity acquisition: innate immune systems spread through microbial populations via horizontal gene transfer (HGT) whereas adaptive-type immune systems acquire immunity via direct interaction with the virus. We developed a mathematical model to explore the short term evolution of prokaryotic immunity and show that in stable environments with predictable viral repertoires, adaptive-type immunity is the optimal defense strategy whereas in fluctuating environments with unpredictable virus composition, HGT dominates the immune landscape.

The molecular mechanisms that enable memories to persist over long time-scales from days to weeks and months are still poorly understood. To develop insights we created a behavioral task where, by varying the frequency of learned associations, mice formed multiple memories but only consolidated some, while forgetting others, over the span of weeks. We then monitored circuit-specific molecular programs that diverge between consolidated and forgotten memories. We identified multiple distinct waves of transcription, i.e., cellular macrostates, specifically in the thalamo-cortical circuit, that defined memory persistence. Notably, a small set of transcriptional regulators orchestrated broad molecular programs that enabled entry into these macrostates. Targeted CRISPR-knockout studies revealed that while these transcriptional regulators had no effects on memory formation, they had prominent, causal, and strikingly time-dependent roles in memory stabilization. In particular, the calmodulin-dependent transcription factor Camta1 was required for initial memory maintenance over days, while Tcf4 and the histone methyl-transferase Ash1l were required later to maintain memory over weeks. These results identify a critical Camta1-Tcf4-Ash1l thalamo-cortical transcriptional cascade required for memory stabilization, and puts forth a model where the sequential, multi-step, recruitment of circuit-specific transcriptional programs enable memory maintenance over progressively longer time-scales.

ABSTRACT The placenta is an important producer of hormones essential for fetal development. Insulin-like growth factor 1 (IGF1) is a hormone primarily produced in the placenta in utero and is an important regulator of various developmental pathways including those in heart and liver. Embryonic disruptions in these developmental pathways can lead to lifelong changes and are often associated with chronic disease. Further, the placenta has sex-specific impacts on offspring development in response to hormonal changes. Previous work has shown that altered expression of Igf1 in the placenta results in sexually dimorphic changes to placental and fetal developmental outcomes. Here, mice underwent placental-targeted CRISPR manipulation for overexpression or partial knockout of Igf1 . At the time of euthanasia, heart and liver tissues were collected and weighed. This dataset presents the heart and liver mass of these postnatal mice. There was a significant increase in proportional heart mass in placental Igf1 overexpression adult female mice and a trending increase in proportional liver mass in placental Igf1 overexpression adult male mice. No significant changes in heart or liver mass were seen in placental Igf1 partial knockout mice. These data provide insight into the impact of placental IGF1 on long-term heart and liver development. VALUE OF THE DATA There is significant evidence for the role of early genetic changes in influencing long-term health outcomes, as laid out by the Developmental Origins of Health and Disease (DOHaD) hypothesis [1]. According to this hypothesis, genetic factors may be critical in determining the timing and severity of chronic disease, with varying effects based on sex. Genetics of the placenta, which makes up the maternal-fetal interface, plays an important role in modulating exposures associated with the DOHaD hypothesis [2]. The placenta provides essential hormones to the fetus during pregnancy [3]. Placental changes are associated with the development of chronic disease and metabolic changes [4,5]. Disruptions in placental functions have been linked to defects including congenital heart disease which affects approximately 40,000 babies each year in the United States [6,7]. The placenta is also linked to metabolic diseases later in life such as nonalcoholic fatty liver disease, a chronic liver disease which has increased in prevalence by over 50% from 1990 to 2019 [5,8,9]. Insulin-like growth factor 1 (IGF1) is a placentally produced factor that regulates pathways involved in fetal growth and development and has been shown to be critical in growth of the heart and liver [10-13]. Despite the importance of the placenta and IGF1 in heart and liver growth, specific links between placental Igf1 expression and developmental outcomes remain understudied. Placental function is known to have sex-specific impacts on fetal growth [14]. Further, Igf1 expression in the placenta is linked to differences in offspring developmental outcomes by sex [15]. Placental Igf1 overexpression and knockout affects offspring in a sexually dimorphic manner. IGF1 is a hormone and interacts with sex hormones, likely contributing to sex differences in response to changes in Igf1 expression [16]. Further research, including the work done to produce this dataset, may help clarify the role of placenta Igf1 expression in fetal outcomes, specifically regarding sex differences. The data presented in this paper provide insight into the effects of placental Insulin-like growth factor 1 overexpression and partial knockout on adult heart and liver mass. More research is needed to understand specific functional impacts on these organs. Further, understanding the effects of placental genetic changes may support the development of future treatments and therapies for placental insufficiencies.

Aerobic methanotrophic bacteria are the primary organisms that consume atmospheric methane (CH 4 ) and have potential to mitigate the climate-active gas. However, a limited understanding of the genetic determinants of methanotrophy hinders the development of biotechnologies leveraging these unique microbes. Here, we developed and optimized a methanotroph CRISPR interference (CRISPRi) system to enable functional genomic screening. We built a genome-wide single guide RNA (sgRNA) library in the industrial methanotroph, Methylococcus capsulatus , consisting of ∼45,000 unique sgRNAs mediating inducible, CRISPRi-dependent transcriptional repression. A selective screen during growth on CH 4 identified 233 genes whose transcription repression resulted in a fitness defect and repression of 13 genes associated with a fitness advantage. Enrichment analysis of the 233 putative essential genes linked many of the encoded proteins with critical cellular processes like ribosome biosynthesis, translation, transcription, and other central biosynthetic metabolism, highlighting the utility of CRISPRi for functional genetic screening in methanotrophs, including the identification of novel essential genes. M. capsulatus growth was inhibited when the CRISPRi system was used to individually target genes identified in the screen, validating their essentiality for methanotrophic growth. Collectively, our results show that the CRISPRi system and sgRNA library developed here can be used for facile gene-function analyses and genomic screening to identify novel genetic determinants of methanotrophy. These CRISPRi screening methodologies can also be applied to high-throughput engineering approaches for isolation of improved methanotroph biocatalysts.

Targeted protein degradation (TPD) has emerged as a highly promising therapeutic strategy for a wide range of diseases, including cancer and neurodegenerative disorders. The ubiquitin-proteasome system, which is responsible for protein degradation, plays a critical role in this process. Gaining comprehensive insights into the ubiquitylation landscape is essential for the development of selective and efficient targeted protein degradation approaches. Recently, data-independent acquisition (DIA) has gained significant popularity as a robust and unbiased approach for quantitative proteomics. Here, we report a cutting-edge workflow that utilizes diGly antibody-based enrichment followed by an optimized Orbitrap-based DIA method for the identification of ubiquitylated peptides. We identify over 40,000 diGly precursors corresponding to more than 7,000 proteins in a single measurement from cells exposed to a proteasome inhibitor, highlighting an exceptional throughput. By applying our optimized workflow, we successfully identify ubiquitylation sites on substrate proteins with various TPD approaches. Therefore, our workflow holds tremendous potential for rapidly establishing mode of action for various TPD modalities, including PROTACs and molecular glues.

The autophagy-tethering factor, ectopic P-granule 5 autophagy protein (EPG5), plays a key role in autophagosome-lysosome fusion. Impaired autophagy associated with pathogenic variants in EPG5 cause a rare devastating multisystem disorder known as Vici syndrome, which includes neurodevelopmental defects, severe progressive neurodegeneration and immunodeficiency. The pathophysiological mechanisms driving disease presentation and progression are not understood. In patient-derived fibroblasts and iPS cells differentiated to cortical neurons, we found that impaired mitophagy leads to mitochondrial bioenergetic dysfunction. Physiological Ca 2+ signals resulted in paradoxical mitochondrial Ca 2+ overload attributed to downregulation of MICU1/3. Ca 2+ signals caused mitochondrial depolarisation, mtDNA release and activation of the cGAS-STING pathway, reversed by pharmacological inhibition of the mitochondrial permeability transition pore (mPTP) or of the STING pathway. Thus, we have identified multiple potential therapeutic targets driving disease progression associated with pathogenic EPG5 mutations, including impaired mitochondrial bioenergetics, mitochondrial Ca 2+ overload, vulnerability to mPTP opening and activation of innate immune signalling.

BACKGROUND Internal N 7 -methylguanosine (m 7 G) is a recently identified chemical modification of mammalian mRNA and a component of the epitranscriptome. While the epitranscriptome plays a key role in regulating RNA metabolism and cellular function, the specific contribution of internal m 7 G to cardiovascular health and disease remains unknown. Atherosclerosis preferentially develops at sites where disturbed blood flow activates endothelial cells, but whether internal m 7 G and its regulatory machinery influence endothelial mechanotransduction and atherogenesis is unclear. METHODS We integrated epitranscriptomic profiling, human tissue analysis, genetically modified mouse models, and targeted nanomedicine approaches to investigate the role of Methyltransferase-like protein 7A (METTL7A), a putative internal m 7 G methyltransferase, in regulating the flow-sensitive endothelial transcriptome and atherosclerosis. Vascular endothelial cells were subjected to well-defined athero-protective and athero-prone flow waveforms in vitro and in vivo . METTL7A function was assessed using RNA sequencing (RNA-seq), liquid chromatography-tandem mass spectrometry (LC-MS/MS), crosslinking immunoprecipitation sequencing (CLIP-seq), RNA stability assays, and a CRISPR-Cas-inspired RNA targeting system (CIRTS). METTL7A expression in human coronary arteries with and without atherosclerosis was evaluated by RNA-seq and immunostaining. In vivo atherosclerosis studies were conducted in both global and endothelial-specific Mettl7a1 knockout mice. Endothelial METTL7A expression was restored using cationic polymer-based nanoparticles delivering CDH5 promoter-driven METTL7A plasmids or VCAM1-targeted lipid nanoparticles delivering N1-methylpseudouridine (m¹Ψ)-modified METTL7A mRNA. RESULTS Athero-protective unidirectional flow significantly induced METTL7A expression, which promoted internal m 7 G methylation of endothelial transcripts, while other major epitranscriptomic marks and cap-associated m 7 G were not affected by METTL7A. METTL7A preferentially binds to AG-enriched motifs in protein-coding mRNAs and plays a key role in regulating KLF4 and NFKBIA transcripts, enhancing their internal m 7 G and stability and supporting vascular homeostasis. In contrast, endothelial METTL7A expression was significantly reduced by disturbed blood flow and in human atherosclerotic lesions. Global or endothelial-specific loss of METTL7A exacerbated disturbed flow-induced atherosclerosis in mice, independent of serum lipid levels. Restoration of endothelial METTL7A, via nanoparticle-mediated plasmid or m 1 Ψ mRNA delivery, markedly reduced lesion formation in Mettl7a1 ⁻/⁻ and ApoE ⁻/⁻ mice. CONCLUSIONS These findings establish METTL7A as a previously unrecognized mechanosensitive methyltransferase that maintains endothelial homeostasis by stabilizing key anti-inflammatory transcripts, KLF4 and NFKBIA, through internal m 7 G methylation. Loss of METTL7A disrupts endothelial function and accelerates atherogenesis in response to disturbed flow. Therapeutic restoration of endothelial METTL7A, via targeted nanoparticle-mediated gene or m 1 Ψ mRNA delivery, significantly lessens atherosclerosis. Collectively, these results uncover a novel epitranscriptomic mechanism governing vascular health and position METTL7A as a promising target for precision nanomedicine in atherosclerotic cardiovascular disease.

Optical pooled screening (OPS) has emerged as a powerful technique for functional genomics, enabling researchers to link genetic perturbations with complex cellular morphological phenotypes at unprecedented scale. However, OPS data analysis presents challenges due to massive datasets, complex multi-modal integration requirements, and the absence of standardized frameworks. Here, we present Brieflow, a computational pipeline for end-to-end analysis of fixed-cell optical pooled screening data. We demonstrate Brieflow’s capabilities through reanalysis of a CRISPR-Cas9 screen encompassing 5,072 fitness-conferring genes, processing more than 70 million cells with multiple phenotypic markers. Our analysis reveals functional gene relationships that were missed in the original study, uncovering coherent biological insights related to mitochondrial function, mRNA processing, vesicular trafficking, and MYC transcriptional control, amongst others. The modular design and open-source implementation of Brieflow facilitates the integration of novel analytical components while ensuring computational reproducibility and improved performance for the use of high-content phenotypic screening in biological discovery.

The plant root microbiome is vital in plant health, nutrient uptake, and environmental resilience. To explore and harness this diversity, we present metagRoot, a specialized and enriched database focused on the protein families of the plant root microbiome. MetagRoot integrates metagenomic, metatranscriptomic, and reference genome-derived protein data to characterize 71,091 enriched protein families, each containing at least 100 sequences. These families are annotated with multiple sequence alignments, CRISPR elements, Hidden Markov Models, taxonomic and functional classifications, ecosystem and geolocation metadata, and predicted 3D structures using AlphaFold2. MetagRoot is a powerful tool for decoding the molecular landscape of root-associated microbial communities and advancing microbiome-informed agricultural practices by enriching protein family information with ecological and structural context. The database is available at https://pavlopoulos-lab.org/metagroot/ or https://www.metagroot.org

CRISPR-Cas9-based gene editing is a powerful approach to developing gene and cell therapies for several diseases. Engineering cell therapies requires accurate assessment of gene editing allelism because editing patterns can vary across cells leading to genotypic heterogeneity. This can hinder development of robust cell therapies. Droplet-based targeted single-cell DNA sequencing (scDNAseq) has been used to genotype targeted loci across thousands of cells enabling high-throughput assessment of gene editing efficiency. Here, we constructed a “ground truth” gene editing single-cell DNAseq atlas, along with an artifact-aware computational workflow called GUMM (Genotyping Using Mixture Models) to systematically infer single-cell allelism from these data. This resource was created by expanding CRISPR-Cas9-edited HL-60 clones that harbored distinct insertion-deletion (indel) profiles in CLEC12A and mixing them at pre-defined ratios to create artificial cocktails that mimic the potential editing diversity of a CRISPR-Cas9 experiment. This enabled assessment of technical artifacts that confound interpretation of allelism in the readouts of gene edited cells. GUMM was able to accurately genotype cells and infer the original clonal composition of the artificial cocktails even in the presence of artifacts.

Many cancer types upregulate expression of sialic acid-containing glycans. These oligosaccharides subsequently engage inhibitory Siglec receptors on immune cells, allowing cancer cells to evade immune surveillance. The genetic mechanisms by which this glycome remodeling occurs remain poorly defined. Understanding the ways that cancer cells change their cell surface glycosylation is critical for identification of biomarkers and targets for glycan-directed immunotherapy. In this study, we performed multiple gain-of-function CRISPR activation (CRISPRa) screens to broadly define genetic pathways that regulate expression of Siglec-binding glycans. We show that Siglec ligand expression is largely controlled through genetic competition between genes that catalyze α2-3 sialylation and GlcNAcylation of galactose residues. Perturbation of enzyme expression at this key biosynthetic node provides multiple “paths” by which cancers can acquire elevated expression of Siglec ligands. We further show that cancer glycome remodeling is aided by overexpression of novel “professional ligands” that facilitate Siglec-glycan binding. Notably, we also find that expression of the CD24 gene is genetically dispensable for cell-surface binding of the inhibitory receptor Siglec-10. Finally, by integrating our functional genetic model with clinical tumor genomic data, we identify the sulfotransferase enzyme GAL3ST4 as a potential novel driver of immune evasion in glioma cells. Taken together, this study provides a first-in-class genomic atlas to aid understanding of cancer-associated glycosylation and identifies immediately actionable targets for cancer immunotherapy.

Artemisia annua is renowned for producing artemisinin, a compound that revolutionized malaria treatment and holds therapeutic promise for other diseases, including cancer and diabetes. However, low natural yields of artemisinin remain a major bottleneck, necessitating a deeper understanding of the genetic and regulatory networks involved in its biosynthesis. Although several transcriptomic studies on A. annua exist, they are often limited in scope, and a comprehensive, tissue-resolved gene expression resource has been lacking. Here, we present the Artemisia Database (Artemisia-DB)—a high-resolution expression atlas constructed from an extensive integration of publicly available RNA-seq datasets. The database provides transcript- and gene-level abundance estimates across major tissues and includes functional annotations such as Gene Ontology (GO) terms, KEGG pathways, and InterPro domains. As a case study, we investigated the coexpression profile of HMGR (3-hydroxy-3-methylglutaryl- CoA reductase), a key enzyme in the mevalonate pathway and an early step in artemisinin biosynthesis. Coexpression analysis in leaf tissue revealed a subset of Auxin Response Factor (ARF) transcription factors strongly correlated to HMGR. This finding suggests a potential regulatory link between auxin signaling and artemisinin biosynthesis, providing new hypotheses for functional validation. Artemisia-DB is freely accessible at https://artemisia-db.com and offers an interactive interface for exploring expression data, functional annotations, transcription factors, CRISPR targets, and more. By combining high-quality transcriptome data with regulatory and functional insights, Artemisia-DB serves as a valuable resource for the plant research community and facilitates deeper investigation into the transcriptional dynamics and specialized metabolism of A. annua .

In recent decades, the idea to develop crop varieties which can handle environmental stressors has been taking form. Many effective genetic engineering tools have been developed, but many of those techniques are prone to ecological risks and face challenges from regulatory approvals and public perspectives. So this paper outlines a non-transgenic approach which battles transgenic constraints through a method called SmartNative Genome Editing, in which crop resilience is increased by using its native gene sequences. The main components of SmartNative Genome Editing include Cisgenic Regulatory Editing (CRE) and Epigenetic Priming-Based Trait Modulation (EPTM). Cisgenic regulatory editing (CRE) modifies the native regulatory patterns of plants using CRISPR-Cas9. Epigenetic Priming-Based Trait Modulation (EPTM) involves modification through epigenetic modifiers to mimic the actual environmental stress and simulate stress memory in plants, by which plants can easily respond faster when real conditions prevail. With both CRE and EPTM methods regulating gene expression without inserting foreign DNA will be a safer and more acknowledged pathway for crop improvement and have better environmental adaptability.