Background Mavacamten, a first-in-class allosteric myosin inhibitor, has demonstrated efficacy and safety in obstructive hypertrophic cardiomyopathy (oHCM), notably reducing symptoms, left ventricular outflow obstruction, and wall thickness over 30 weeks. We recently reported that the MYBPC3 c.772G>A variant causes HCM through cMyBP-C haploinsufficiency, leading to accelerated sarcomere kinetics and higher energy consumption in patient myocardium and hiPSC- derived cardiomyocytes (hiPSC-CMs). These effects are counterbalanced by prolonged action potentials and slower Ca²⁺ transients, which preserve twitch duration but may increase arrhythmic risk. Mavacamten may reduce myocardial energetic defects in HCM. Objectives To investigate the long-term effects of Mavacamten on sarcomere structure, contractility, and transcriptional remodeling using patient-specific and CRISPR-corrected isogenic hiPSC-derived cardiomyocyte models of HCM. Methods HiPSC-CMs and engineered heart tissues (EHTs) derived from a MYBPC3:c.772G>A patient and its CRISPR-corrected line were first exposed to increasing concentrations of Mavacamten to assess acute dose–response relationships and determine IC 50 values. Based on these data, chronic treatments (0.3– 0.75 μM for 20 days) were performed mechanical, structural, electrophysiological, and transcriptomic adaptations. Results Acute exposure produced a rapid and fully reversible reduction in active force, while chronic treatment for 20 days induced a sustained decrease in contractility with incomplete recovery after 4 days of washout, indicating a two-phase mechanism of action. Long-term force reduction was paralleled by decreased cell area and sarcomere density, indicating that structural disassembly contributes to sustained functional depression and re-assembly after washout. Electrophysiological analysis confirmed the specific alterations of the MYBPC3 c.772G>A mutation previously observed, with no detectable effects following treatment with Mavacamten. In addition, transcriptome analysis was used to study the molecular mechanisms underlying the long-term effect. Conclusions Mavacamten induces a biphasic, persistent-to-reversible, reduction of sarcomeric force associated with structural remodeling, providing mechanistic insight into its capacity to promote favorable cardiac remodeling in oHCM.

Desminopathies are a heterogeneous group of myofibrillar myopathies defined by the presence of desmin-positive aggregates that compromise cytoskeletal integrity in skeletal and cardiac muscle. Although desmin knockout models and several truncating mutations typically result in a functional null phenotype without inclusion body formation, the molecular consequences of specific stop-gain variants remain poorly understood. In this study, we investigated the pathogenic mechanism of a novel DES nonsense mutation, NM_001927.4:c.448C>T; p.(Arg150Stop), previously identified in an Indian patient with congenital myopathy. This premature stop codon lies within the Linker 1A domain and is predicted to generate a truncated protein lacking the C-terminal tail. To delineate its functional consequences, we used two complementary experimental approaches: transient overexpression of the R150X mutant in skeletal and cardiac myocytes, and a CRISPR/Cas9-engineered homozygous R150X cardiomyocyte line (Des-R150X-CRISPR). Both models consistently revealed the formation of persistent aggregate-like structures, in striking contrast to desmin knockout systems that do not generate inclusions. These aggregate-like structures disrupted actin filament organization, impaired filament bundling, and induced organelle mislocalization. Biochemical analysis indicated that the aggregates were resistant to proteasomal degradation, yet they were partially cleared by autophagy, underscoring a role for protein quality control pathways in modulating disease severity. Importantly, the Des-R150X-CRISPR line demonstrated aggregate-driven pathology at endogenous levels, confirming that this mutation acts through a toxic gain-of-function mechanism rather than simple loss of desmin function. Our findings establish the Arg150Stop variant as a mechanistically distinct truncating mutation that generates aggregation-prone protein rather than a null state. By reproducing hallmark features of desminopathy in a physiologically relevant human cell models, this work not only broadens the known pathogenic spectrum of DES variants but also highlights aggregate formation as a central driver of cellular dysfunction and a promising therapeutic target in desminopathies.

Nanomedicine has now become a radical agent of change in the game of immunotherapy and introduced precision, control, and customization like never before when it comes to cancer, as well as autoimmune conditions. Using platforms based on nano scale, researchers have been able to manipulate immune responses by working on a scale both spatial and temporal in such a way that it helps overcome the drawbacks associated with working with immune response such as immune evasion, systemic toxicity and poor pharmacokinetics. Sophisticated nanoparticles (such as stimuli-sensitive ones, exosome-mimetic vesicle nanoparticles, and nanoparticles with CRISPR) allow directed immunomodulators, antigens and gene-editing systems to reach one or more particular immune compartments. The innovations allow reprogramming of immune cells, immune tolerance rejuvenation and expansion of antitumor immunity without significant off-target effects. Finding applications in integrating the artificial intelligence as well as multi-omics techniques, the process leads to personalization of the nano-immunotherapies based on patient-specific immuno-signatures. The chapter discusses the mechanistic rationale, therapeutic advancement, and the translational opportunities of nanotechnology-based immunotherapies that define them as part of a foundation of future generations of clinical approaches to precision immune modulation in oncology and autoimmune diseases.

Precision oncology is broadly defined as cancer prevention, diagnosis, and treatment specifically tailored to the patient based on his/her genetics and molecular profile. In simple terms, the goal of precision medicine is to deliver the right cancer treatment to the right patient, at the right dose, at the right time. Precision oncology is the most studied and widely applied subarea of precision medicine. Now, precision oncology has expanded to include modern technology (big data, single-cell spatial multiomics, molecular imaging, liquid biopsy, CRISPR gene editing, stem cells, organoids), a deeper understanding of cancer biology (driver cancer genes, single nucleotide polymorphism, cancer initiation, intratumor heterogeneity, tumor microenvironment ecosystem, pan-cancer), cancer stratification (subtyping of traditionally defined cancer types and pan-cancer re-classification based on shared properties across traditionally defined cancer types), clinical applications (cancer prevention, early detection, diagnosis, targeted therapy, minimal residual disease monitoring, managing drug resistance), lifestyle changes (physical activity, smoking, alcohol consumption, sunscreen), cost management, public policy, and more. Despite being the most developed area in precision medicine, precision oncology is still in its early stages and faces multiple challenges that need to be overcome for its successful implementation. In this review, we examine the history, development, and future directions of precision oncology by focusing on emerging technology, novel concepts and principles, molecular cancer stratification, and clinical applications.

The major pathological hallmarks of sporadic and familial forms of Parkinson’s disease (PD) are the targeted and progressive loss of midbrain dopaminergic neurons (mDA), associated with systemic iron accumulation, α-synuclein (αsyn) accumulation and aggregation, and lipid peroxidation amongst other reactive oxygen species (ROS) generation. Therapeutic strategies aimed towards dopamine restoration, αsyn removal and iron chelation have provided symptomatic relief but failed to prevent or slow disease progression. This is in part due to the lack of understanding of the exact pathways leading to neuronal death in PD. In this study, we investigate ferroptosis, a unique cell death mechanism sharing multiple features with PD pathology, as a relevant pathway with implications in disease pathogenesis. We identified an enrichment of ferroptosis genes dysregulated throughout PD postmortem brain samples and several neuronal and glial PD models. Using CRISPR/Cas9 technology, we generated a rapid iPSC-derived synucleinopathy neuronal model harbouring the SNCA A53T mutation and report increased ROS generation, reduced levels of antioxidant glutathione (GSH), impaired mitophagy and a heightened vulnerability to ferroptosis-induced lipid peroxidation and cell death. Critically, inhibition of the key lipid peroxidation enzyme and driver of ferroptosis, 15-lipoxygenase (15-LO), rescued synucleinopathy associated pathologies and prevented pathological αsyn oligomerisation in SNCA A53T neurons. Furthermore, we report enhanced microglial ferroptosis susceptibility in models of synucleinopathy. In summary, we highlight a new mechanism by which the familial PD-associated SNCA A53T mutation causes cell death and propose 15-LO inhibition as a tractable therapeutic opportunity in PD.

Background Antimicrobial resistance (AMR) in Escherichia coli is a critical global health challenge, particularly in urinary tract infections, where first-line treatments are increasingly compromised. While horizontal gene transfer (HGT) via mobile genetic elements is a major driver of AMR, the genomic factors that may constrain resistance gene acquisition remain underexplored. CRISPR-Cas systems, which provide adaptive immunity against foreign DNA, could influence AMR dynamics, but their role in E. coli remains incompletely understood. Methods We conducted a comprehensive whole-genome analysis of uropathogenic E. coli isolates, including a newly sequenced collection from Australian clinical samples and an independent, globally sourced validation cohort. Antimicrobial susceptibility profiles were integrated with CRISPR-Cas subtype classification, resistance gene burden, and mobile element content. Elastic net regression, adaptive lasso, and tree-based machine learning models were used to identify genomic predictors of resistance, with performance validated across both datasets. Results CRISPR-Cas subtype I-F was consistently associated with susceptibility to antibiotics commonly acquired through HGT, including trimethoprim and ampicillin, and linked to lower ARG and MGE burden. In contrast, Type I-E arrays, especially when co-occurring with orphan I-F arrays, were associated with increased resistance. These associations remained robust after adjusting for phylogroup, plasmid content, and genomic background, and were validated across datasets. Conclusions Subtype-specific CRISPR-Cas systems shape antibiotic resistance profiles in E. coli , with Type I-F functioning as a potential genomic barrier to ARG acquisition. These findings highlight CRISPR array typing as a novel biomarker for AMR risk prediction and surveillance, and suggest new opportunities for leveraging CRISPR-based mechanisms to limit resistance propagation in clinical contexts.

Abstract Horizontal gene transfer (HGT) is a major driver of microbial evolution, yet the influence of host cellular context on the integration and functionality of transferred genes remains underexplored. In this study, we investigate how host background affects the compatibility and consequences of acquiring post-translational modification (PTM) machinery through HGT using the heterologous expression of the highly conserved translational elongation factor P (EF-P) from diverse species in Escherichia coli as a model. EF-P and its PTM machinery have been horizontally transferred many times across the bacterial tree of life, and these experiments are meant to examine the consequences of these events. EF-P has a diverse and heterogenous relationship with PTMs; three characterized variants each undergo distinct PTM pathways, while others function effectively without any modification. In this study, we demonstrate that EF-P from Deinococcus radiodurans , Geoalkalibacter ferrihydriticus , and Nitrosomonas communis can complement an EF-P knockout in E. coli without requiring modification, suggesting they represent new examples of unmodified EF-Ps. We also found that the EF-P from the Thermotogota Mesotoga prima is post-translationally modified in an off-target reaction by the rhamnosylation enzyme EarP, thus interfering with its functionality. Conversely, we saw that rhamnosylation by EarP is fully compatible with the EF-P-like protein EfpL from Escherichia coli , thus presenting a promising opportunity to develop novel, catalytically active PTMs. These findings highlight that PTM systems introduced via HGT can have unintended effects on host proteins, emphasizing the complexity of gene integration and functional compatibility in foreign genomic contexts.

Abstract Among microbially derived metabolites that influence host disease, colibactin garners increasing attention for its roles in the rising incidence of early-onset colorectal cancer. Produced by pks⁺ Escherichia coli, colibactin is a potent genotoxin, yet no approved therapeutics directly suppress it. Here, we engineered a self-transmissible conjugative plasmid to deliver CRISPR interference (CRISPRi) into multiple pks⁺ strains. This system silences transcription of colibactin biosynthetic genes and abolishes pks⁺ E. coli genotoxicity without the resistance mutations associated with wild-type Cas9-mediated bacterial inhibition. In mice, conjugation-mediated CRISPRi reduces DNA damage and pks⁺ E. coli colonization while preserving commensal diversity. Importantly, the system also lowers tumorigenesis driven by pks⁺ E. coli and outperforms a pharmacologic inhibitor in a mouse colorectal cancer model. Finally, we extend this platform to silence a second pathogenic metabolite, establishing a translational strategy to neutralize diverse microbial metabolites and expanding the toolkit for programmable live biotherapeutics in the gut.

Abstract The transition from well-fed to food-deprived conditions in C. elegans triggers a stereotypic exploration behaviour in C. elegans characterised by a temporal decrease in cumulative reorientations. We conducted a screen of neuropeptide mutants and identified several candidates involved in modulating this behaviour. Among these, the neuropeptide FLP-15 emerged as a key regulator. Our observations revealed that FLP-15 regulates the frequency of reversals during foraging through the I2 pharyngeal neuron via the G-protein coupled receptor NPR-3. Mutants lacking either flp-15, npr-3 or both displayed a significant defect in reversal frequency which did not decline temporally unlike in wild-type animals. This study also describes the expression pattern of NPR-3 in a subset of head neurons, predominantly comprising of dopaminergic neurons. Finally, flp-15 expression studies and exogenous dopamine supplementation assays revealed that FLP-15 may regulate exploratory search by modulating dopamine transmission, highlighting a novel neuropeptide-dopamine interaction involved in the control of foraging behaviours.

Staphylococcus aureus remains a major clinical threat due to rising antibiotic resistance and high rates of treatment failure. Deciphering the genetic responses to antibiotic pressure and identifying conserved vulnerabilities are essential steps toward developing broadly effective therapies. Here, we constructed strain-resolved CRISPR interference (CRISPRi) libraries targeting all genes in four clinically relevant S. aureus strains spanning major clonal complexes. CRISPRi-seq screens enabled high-resolution mapping of their fitness landscapes and the definition of a core essentialome representing robust targets for antimicrobial intervention. Exposure of the CRISPRi libraries to four mechanistically distinct antibiotics revealed genome-wide susceptibility profiles, identifying both strain-dependent and conserved susceptibility signatures shaped by the drug mode of action and genetic background. Analysis of these conserved vulnerabilities provided insight into antibiotic-specific stress responses and resistance mechanisms. Among the core determinants of vancomycin vulnerability, we identified several previously uncharacterized genes, including a conserved membrane-associated operon, here designated EsrABC, whose disruption markedly increases vancomycin sensitivity in the four strains. Our study provides a genome-wide atlas of S. aureus fitness and conditional vulnerabilities, fully explorable in the here-developed online AureoBrowse platform ( https://aureobrowse.veeninglab.com/ ), revealing candidates for synergistic therapies and potential therapeutic targets.

Autophagy (cellular self-eating) is a tightly regulated catabolic process of eukaryotic cells in which parts of the cytoplasm are sequestered and subsequently degraded within lysosomes by acidic hydrolases. This process is central to maintaining cellular homeostasis, the removal of aged or damaged organelles, and the elimination of intracellular pathogens. The nematode Caenorhabditis elegans has proven to be a powerful genetic model for investigating autophagy. To date, the fluorescent autophagy reporters developed in this organism have predominantly relied on multi-copy, randomly integrated transgenes. As a result, the interpretation of autophagy dynamics in these models has required considerable caution due to possible overexpression artifacts and positional effects. Here, we describe the development of two endogenous autophagy reporters, engineered using CRISPR-Cas9 genome editing: gfp::mCherry::lgg-1/atg-8 and gfp::atg-5, both inserted precisely into their endogenous genomic loci. These single-copy reporters reliably track distinct stages of the autophagic process. Using these tools, we demonstrate that (i) the transition from the earliest phagophore to the mature autolysosome is an exceptionally rapid event, (ii) starvation triggers autophagy only after a measurable lag phase, rather than immediately, and (iii) autophagy in C. elegans is subject to strict regulatory control, preventing excessive flux that could otherwise compromise cellular survival. We anticipate that these newly developed reporter strains will provide refined opportunities to dissect the physiological and pathological roles of autophagy in vivo.

We present a platform that directly sequences single guide RNAs and endogenous 3′UTRs in fixed cells while simultaneously measuring protein abundance and cellular morphology. We demonstrate platform capability by performing optical pooled screening of CRISPR-perturbed lung cancer cells. This approach unites direct in-sample RNA sequencing with complementary phenotypic readouts, enabling comprehensive, scalable, and functional genomics analyses within a single experiment.

Attaching effacing (A/E) bacteria, such as Enteropathogenic E. coli (EPEC) and Citrobacter rodentium , colonize intestinal epithelial cells (IECs) by inducing remodeling of the epithelial cytoskeleton and formation of prominent actin pedestals at bacterial attachment sites. While non-muscle myosin II (NM II) is a key regulator of the actin cytoskeleton, whether it regulates IEC colonization by A/E pathogens is not known. To address this question, we targeted NM IIA and NM IIC, the NM II paralogs expressed in IECs. Our in vivo studies utilized mouse models with either intestinal epithelial-specific deletion of NM IIA (NM IIA cKO mice), expression of a NM IIA motor domain mutant, or total deletion of NM IIC (NM IIC tKO mice). In vitro experiments utilized IECs (HT-29cF8 and Caco-2BBE) with CRISPR-Cas9-mediated deletion of NM IIA or NM IIC. In addition, NM II activity in vitro was modulated pharmacologically, using either the pan-myosin inhibitor, blebbistatin, or a specific NM IIC activator, 4-hydroxyacetophenone (4-HAP). NM IIA cKO and NM IIA mutant mice demonstrated higher C. rodentium colonization along with more severe mucosal inflammation and colonic crypt hyperplasia as compared to their controls. By contrast, NM IIC tKO mice was indistinguishable from their control with regard to C. rodentium colonization. Blebbistatin treatment increased EPEC attachment to IECs monolayers, whereas 4-HAP did not affect bacterial attachment. Genetic knockout of NM IIA, but not NM IIC, increased EPEC adhesion to IEC monolayers. Importantly, the increase in EPEC attachment exhibited by NM IIA-deficient IECs required intact bacterial Type 3 secretion system and functional Tir effector, indicating that NM IIA functions in actin pedestal assembly. In summary, we describe a novel role for NM IIA in limiting intestinal epithelial colonization by A/E pathogens via inhibition of pathogen-induced remodeling of the actin cytoskeleton.

Summary Microglia, the brain’s innate immune cells, can adopt a wide variety of activation states relevant to health and disease. Dysregulation of microglial activation occurs in numerous brain disorders, and driving or inhibiting specific states could be therapeutic. To discover regulators of microglial activation states, we conducted CRISPR interference screens in iPSC-derived microglia for inhibitors and activators of six microglial states. We identified transcriptional regulators for each of these states and characterized 31 regulators at the single-cell transcriptomic and cell-surface proteome level in two distinct iPSC-derived microglia models. Finally, we functionally characterized several regulators. STAT2 knockdown inhibits interferon response and lysosomal function. PRDM1 knockdown drives disease-associated and lipid-rich signatures and enhanced phagocytosis. DNMT1 knockdown results in widespread loss of methylation, activating negative regulators of interferon signaling. These findings provide a framework to direct microglial activation to selectively enrich microglial activation states, define their functional outputs, and inform future therapies. Highlights CRISPRi screening reveals novel regulators of six microglia activation states Multi-modal single-cell screens highlight differences between mRNA and protein level expression iPSC-microglia models show different baseline distributions of activation states Loss of DNMT1 leads to widespread DNA demethylation, promoting some states but limiting the interferon-response state Loss of PRDM1 drives microglial disease-associated state

In early 2025, Sierra Leone experienced a large outbreak of mpox clade IIb, underscoring the urgent need for portable, low-cost diagnostics in decentralized settings. We rapidly developed and field-deployed Mpox SHINE, a CRISPR–Cas13 assay that integrates lyophilized reagents, ambient-temperature lysis, and automated fluorescence detection on a portable device, the DxHub. The assay achieved analytical sensitivity down to 10 copies/µL with minimal hands-on time. In-country evaluation of 56 clinical specimens showed complete concordance with qPCR, with 100% sensitivity (45/45) and 100% specificity (11/11) at the sample level. Mpox SHINE also detected the virus directly from unextracted lesion swabs, maintaining 100% sensitivity and specificity in 16 samples (8 positive, 8 negative). Across extracted and unextracted samples, the mean time-to-result was ∼35 minutes, with all positives detected within 45 minutes. Thus, Mpox SHINE performed on the DxHub demonstrates how CRISPR-based pathogen detection can be rapidly translated into portable tools for the front lines of outbreak response. Teaser CRISPR-Cas13 assay and portable device bring rapid, reliable mpox testing to outbreak settings.

X-linked Dystonia-Parkinsonism (XDP) is a lethal adult-onset neurodegenerative disorder that exhibits features of dystonia and parkinsonism and is exclusively associated with a causal founder haplotype that is indigenous to the Philippines and affects Filipino males. Using patient-specific fibroblasts, neural stem cells (NSC), and other neuronal models, we discovered that cryptic alternative splicing caused by a novel SINE-VNTR-Alu (SVA) mobile element insertion into intron 32 of TAF1 is a mechanistic hallmark of XDP. We leveraged postmortem brain samples from an XDP-specific brain bank to demonstrate that the molecular hallmarks of XDP observed in neural stem cells (NSCs) mirror abnormalities observed in brain tissues from affected patients. Based on these findings that patient-specific NSCs reproduce mechanistic signatures found in the brain, we sought to develop a bespoke precision therapeutic for XDP and evaluate its relative efficacy in ameliorating transcriptomic signatures in neuronal models. We first used CRISPR-based excision of the SVA and demonstrated ablation of all aberrant splicing and dysregulation of TAF1 expression in NSCs across 30 independent clones. CRISPR-based correction of the XDP haplotype also restored the expression of 424 of 1,490 (30%) differentially expressed genes (DEGs) that were altered in XDP patient lines and greatly exceeded what would be expected by chance (p-value = 9.89e-87). While in vivo delivery of a gold standard CRISPR therapy is currently not feasible for XDP, we evaluated a tractable approach for Filipino patients by exploring the potential to modulate alternative splicing in XDP patients using antisense oligonucleotides (ASOs). To accomplish this, we developed a large-scale and well controlled functional genomics platform that screened eighty ASOs targeting intron 32 of XDP patients, followed by prioritization of lead ASOs based on attenuation of the alternative splicing signature. In transcriptome analyses across 1,550 libraries, we found that 8 of the 12 lead ASOs ameliorated the targeted XDP aberrant splicing. Moreover, we found that the two lead ASOs exhibited 38% and 43% rescue of XDP-specific DEGs that were also rescued by CRISPR excision of the SVA (enrichment p-values = 2.06e-13 and 2.27e-05, respectively). These rescues represented restoration of key molecular functions previously implicated in XDP, such as synaptic function, DNA-binding transcription factor activity, and gliogenesis. This study highlights a path to a potential targeted therapeutic for XDP and the capacity to exploit functional genomic signatures in patient-derived neural models to develop a scalable precision therapeutic platform for rare genetic disorders.

Summary Modern molecular biology tools and technologies such as CRISPR have sped up scientific discovery. From an educational perspective, these advancements are both exciting and overwhelming. Educators shaping these future scientists face the ongoing challenge of staying compliant with the latest developments in molecular biology while finding effective ways to teach these discoveries. As the use of CRISPR gene editing technology continues to expand globally, there is an increasing need for a workforce that is both knowledgeable about its theoretical foundations and trained in its practical use. While advanced, technology-driven STEM courses have the potential to improve student retention, they are often lecture-heavy and lack intentional engagement strategies that support deeper learning. Moreover, agriculture is the second most impacted sector by this technology, yet there is a significant lack of teaching materials focused on CRISPR in plant biology. To address these gaps, we developed a framework for teaching gene editing that incorporates multiple engagement strategies beyond traditional lecture-based instruction. This framework was implemented over two semesters in an Introduction: to Gene Editing course at Tennessee State University, offered to both undergraduate and graduate students enrolled in a degree in Agricultural Sciences. This manuscript outlines the various strategies used in the course including active learning, multimodal instructional approaches and experiential learning strategies that can be adopted in other classrooms to effectively teach gene editing. Survey-based results from the course indicate a measurable increase in student comfort with designing and executing CRISPR-Cas based experiments. Societal Impact Statement Plant biology lacks accessible teaching materials for CRISPR, a powerful gene-editing technology widely used to improve agriculture. We developed an engaging framework to teach CRISPR concepts to advanced undergraduates and graduate students in plant sciences, which can be readily adopted by other instructors. The approach increased self-reported confidence in students and comfort in explaining CRISPR. Since instructors often have limited time to design interactive lessons, this framework offers a ready-to-use, effective strategy that makes CRISPR more widely available in classrooms, ultimately strengthening CRISPR literacy in the future agricultural workforce.

ABSTRACT Diatoms are major phytoplanktonic algae with secondary endosymbiotic plastids that differ in cellular and regulatory traits from those of the green lineage. Here we exploited the heterotrophic growth ability of Cyclotella cryptica to create the first diatom photosynthetic mutant by CRISPR-Cas inactivation of the nucleus-encoded ATP synthase subunit γ. These mutants showed impaired phototrophic capacity and altered thylakoids morphology. In absence of γ-ATP, protons that accumulate in the thylakoid lumen slow down the cytochrome b 6 f complex, thus keeping the electron carriers downhill oxidized. These results, reversible when the proton gradient is suppressed, demonstrate the existence of a photosynthetic control in diatoms. At variance with the wild type, γ ATP synthase mutants cannot grow heterotrophically in darkness nor in the light when photosystem II is inhibited. This requirement of heterotrophic growth on photosynthetic electron transfer or on the presence of plastidial ATP synthase suggests that the proton motive force (pmf) is a central integrator of the metabolic interaction between photosynthesis and heterotrophy. Our results establish C. crytica as a robust model for analysis of photosynthetic function, regulation and metabolic integration in organisms with secondary plastids. Teaser Mutagenesis in the facultative autotroph diatom Cyclotella cryptica enables exploration of essential plastid functions in diatoms

ABSTRACT Neuronal loss is a hallmark of neurodegeneration and brain injury. Direct reprogramming of astrocytes into neurons has emerged as a promising approach to restore lost neurons. Comprehensive mapping and characterization of candidate astrocyte-to-neuron reprogramming factors is an essential step to realizing the potential of this strategy. Here, we established a CRISPR activation (CRISPRa)-based approach for neuronal reprogramming of primary human astrocytes. We conducted high-throughput CRISPRa screens of all human genes encoding transcription factors (TFs) to identify novel and efficient reprogramming factors. scRNA-seq characterization of top hits revealed that single TFs reprogram primary human astrocytes into multiple neuronal subtypes with distinct cell type-specific gene signatures. We demonstrate that INSM1 reprograms astrocytes to a glutamatergic neuron-like state and has broad neurogenic activity across different cell types and across human and mouse contexts. Finally, we conduct paired CRISPRa screens to identify cofactors that cooperate with INSM1 to enhance neuronal reprogramming and subtype specification, and elucidate genomic mechanisms of interaction and downstream regulators.

Variants in the LRRK2 and GBA1 genes are among the most common risk factors associated with Parkinson’s disease (PD). Both patients carrying PD-associated variants in GBA1 , encoding lysosomal enzyme glucocerebrosidase (GCase), and a subset of non-carrier patients have been shown to have reduced GCase enzymatic activity, suggesting that reduced GCase activity may be a feature of both genetic and a subset of sporadic PD. However, the effect of PD-associated variants in LRRK2 , encoding a serine/threonine kinase, on GCase activity remains controversial, with conflicting results in various tissues and cell types. Moreover, rare patients carrying both GBA1 and LRRK2 risk alleles seem to have a more benign disease course than carriers of GBA1 variants alone, suggesting a complex interplay between these two genes in PD. Here we evaluate the effect of LRRK2 kinase activity on GCase activity in human induced pluripotent stem cell (iPSC)-derived microglia (iMGs), a PD-relevant brain cell type expressing high levels of LRRK2. Using CRISPR editing, isogenic control iPSC lines were generated to match PD patient-derived iPSC lines harbouring the LRRK2 p.G2019S, p.M1646T, or p.N551K-p.R1398H protective haplotype variants. Whereas iMGs harbouring the p.M1646T variant, and the protective haplotype, respectively increased and decreased phosphorylation of canonical LRRK2 substrate, Rab10, GCase protein levels and activity were not altered in any of the LRRK2 variant lines. Additionally, whereas pharmacological inhibition of LRRK2 kinase activity had no impact on GCase activity in iMGs under basal conditions, it attenuated the increase in GCase activity elicited in response to interferon γ (IFNγ) treatment. Moreover, GCase activity induced by IFNγ was reduced in PD risk LRRK2 p.M1646T iMGs and increased in p.N551K-p.R1398H protective haplotype iMGs compared to their isogenic corrected controls, congruent with their respective effects on LRRK2 kinase activity and PD risk. Thus, our data suggests a role for LRRK2 kinase activity in regulation of GCase activity in response to neuroinflammation.

SUMMARY We map human artery and vein endothelial cell (EC) differentiation from pluripotent stem cells, and employ this roadmap to discover new mechanisms of vascular development (vein differentiation) and disease (viral infection). We discovered vein development unfolds in two steps driven by opposing signals: VEGF differentiates mesoderm into “pre-vein” ECs, but surprisingly, VEGF/ERK inhibition subsequently specifies vein ECs. Pre-vein ECs co-expressed certain arterial ( SOX17 ) and venous ( APLNR ) markers, harbored poised chromatin at future venous genes, but completed venous differentiation only upon VEGF inhibition. Intersectional lineage tracing revealed that early Sox17 + Aplnr + ECs also formed veins in vivo . Next, we compared how Ebola, Andes, and Nipah viruses infect artery and vein ECs under biosafety-level-4 containment. Each virus distinctly affected ECs. Interestingly, artery and vein ECs also responded divergently to the same virus, thus revealing that developmentally-specified cell identity impacts viral infection. Collectively, this arteriovenous differentiation roadmap illuminates vascular development and disease.

ABSTRACT Extrachromosomal DNA (ecDNA) is a prevalent and devastating form of oncogene amplification in cancer 1,2 . Circular megabase-sized ecDNAs lack centromeres and segregate stochastically during cell division 3–6 yet persist over many generations. EcDNAs were first observed to hitchhike on mitotic chromosomes into daughter cell nuclei over 40 years ago with unknown mechanism 3,7 . Here we identify a family of human genomic elements, termed retention elements, that tether episomes to mitotic chromosomes to increase ecDNA transmission to daughter cells. We develop Retain-seq, a genome-scale assay that reveals thousands of human retention elements conferring generational persistence to heterologous episomes. Retention elements comprise a select set of CpG-rich gene promoters and act additively. Live-cell imaging and chromatin conformation capture show that retention elements physically interact with mitotic chromosomes at regions which are mitotically bookmarked by transcription factors and chromatin proteins, intermolecularly recapitulating promoter-enhancer interactions. Multiple retention elements are co-amplified with oncogenes on individual ecDNAs in human cancers and shape their sizes and structures. CpG-rich retention elements are focally hypomethylated; targeted cytosine methylation abrogates retention activity and leads to ecDNA loss, suggesting that methylation-sensitive interactions modulate episomal DNA retention. These results highlight the DNA elements and regulatory logic of mitotic ecDNA retention. Amplifications of retention elements promote the maintenance of oncogenic ecDNA across generations of cancer cells, revealing the principles of episome immortality intrinsic to the human genome.

The C1858T PTPN22 (R620W) variant has been implicated in the pathogenesis of several autoimmune disorders and represents a promising immunotherapeutic target for Type 1 diabetes. We have been implementing a novel immunotherapeutic approach based on the use of lipoplexes that deliver siRNA duplexes. Efficacy and safety of lipoplexes halting variant expression was demonstrated in the peripheral blood of patients in vitro. According to regulatory authorities in Europe preclinical safety and efficacy must be ascertained in vivo in appropriate animal models before undertaking clinical investigations. In the light of the foregoing the aim of this study was to verify that lipoplexes against the murine Ptpn22-R619W, equivalent to the human PTPN22-R620W, could be used for animal experimentation. The murine fibroblast cell line L929 was transfected with the PF62-pLentiPtpn22-R619W plasmid. We designed siRNA duplexes specific for the Ptpn22-R619W allele and formulated them into cationic lipoplexes in order to halt the variant expression in the transfected L929 cell line. Transfection of fibroblasts expressing R619W using lipoplexes resulted in efficient silencing at 100 pmol siRNA after 48 hours post-transfection reaching higher significant knock‑down after 72 hours. Lipoplexes efficiently suppress the pathogenic Ptpn22 variant expression in vitro, supporting the feasibility of a pre‑clinical platform for in vivo lipoplexes testing in CRISPR‑engineered NOD/ShiLtJ mice carrying the R619W mutation.

Abstract Background Vitamin K 2 (VK 2 ), as a derivative of the menaquinone family, plays an important role in the prevention of osteoporosis and cardiovascular calcification. The realization of the industrialization of VK 2 and the reduction of its production cost have become the focus of attention. Results In this work, an E. coli strain with high VK2 accumulation was constructed through rational metabolic engineering and stepwise improvement based on regulatory metabolic information and CRISPR/Cas9-mediated gene knockout. We first constructed a recombinant E. coli to produce menaquinol-8 (MKH 2 -8, a reduced form of VK 2 ) by overexpressing menA and ubiE , the rate-limiting enzymes of the menaquinol pathway. Secondly, we overexpressed different related genes wrbA , qorB and menF , respectively. Among these recombinant strains, the strain MUW reached a yield of 301.96 mg/L after 48 h of fermentation. The optimization of the medium led to an increase in the accumulation of VK 2 . Subsequently, the rational metabolic engineering of gene knockout further increased the VK 2 yield. The recombinant strain ΔB:MUW was selected as the dominant strain for further optimization, with a high VK 2 yield of 723.59 mg/L. A final attempt is to overexpress ispB gene to increased flux of isoprenoid side chain synthesis. After 48 h cultivation, a high VK 2 yield of 1355.29 mg/L was achieved by ΔB:MUWI in a 5 L fermenter. Conclusions This study demonstrates that metabolic engineering techniques combining rational modification of the metabolic pathway and optimization of gene expression can effectively cultivate strains with industrial competitiveness.

We have recently found that by promoting transcriptional elongation, histone deacetylase inhibitors (HDACi) cooperate with the antisense oligonucleotide nusinersen (ASO1) to upregulate exon 7 (E7) inclusion into SMN2 mRNA (Marasco et al., 2022). In parallel, ASO1 also elicits the deployment of the silencing histone mark H3K9me2 on the SMN2 gene, creating a roadblock to RNAPII elongation that downregulates E7 inclusion. By removing the roadblock, HDACi counteract the undesired chromatin effects of the ASO, resulting in higher E7 inclusion, which allowed us to propose a combined therapy for spinal muscular atrophy (SMA). We show here that the histone methyl transferase G9a is involved in the ASO1-elicited deployment of H3K9me2 not only at the SMN2 E7 region, located towards the 3’ end of the gene, but also at its promoter. Furthermore, using a CRISPR/dCas-based (dead-Cas9-based) strategy, we show that targeting H3K27 histone acetylation at the SMN2 E7 region duplicates the HDACi effect, which overcomes potentially pleiotropic effects. Most noticeably, when acetylation is targeted to the E7 region alone, the 30-kbp-distant promoter becomes acetylated and activated. These cross-talks between the two ends of the SMN2 gene prompted us to use chromosome conformation analysis (3C) to find that ASOs can promote gene looping. This novel property of ASOs depends on cohesin and may explain promoter activation by distant alternative splicing events. Abstract Figure In brief Antisense oligonucleotides (ASOs) modulate alternative splicing through base-pairing to sequences in the pre-mRNA. Simultaneously, they may promote the deployment H3K9me2 marks (purple diamons) and looping between the promoter and the 3’end region of a gene, with subsequent transcriptional activation.