ABSTRACT Pennycress ( Thlaspi arvense ) is a winter oilseed domesticated recently to be incorporated as an intermediate crop between the existing cropping systems of the US Midwest. We show that a natural accession of pennycress, 2032, is more susceptible to the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Alternaria japonica than the reference pennycress accession MN106. A previously identified marker associated with early flowering and maturity in pennycress was found to be present in a gene homologous to Arabidopsis Jumonji 14 (JMJ14). It has been reported that AtJMJ14 promotes disease resistance and represses flowering, and greenhouse studies of breeding populations confirmed this phenomenon in pennycress. Plants with the 2032 TaJMJ14 allele were more susceptible to fungi and flowered early. CRISPR-Cas9 editing was used to generate additional TaJMJ14 alleles. A 9-base pair deletion in the 6 th exon of TaJMJ14 showed trends of early flowering and S. sclerotiorum susceptibility, whereas a complete loss-of-function allele led to infertility. We further investigated the transcriptomes of MN106 and 2032 plants in the early stages of S. sclerotiorum and A. japonica infection to identify potential resistance and susceptibility genes. Differences in the expression of pathogen-associated molecular pattern-triggered immunity (PTI)-associated genes led us to discover that 2032 plants have defects in elicitor-triggered oxidative bursts. The transcriptional responses unique to each accession lay a foundation for future gene-editing and breeding approaches to keep the beneficial early flowering phenotype conferred by 2032 but uncouple it from disease susceptibility.

Insects, such as Drosophila melanogaster, rely on innate immune defenses to combat microbial threats. Antimicrobial peptides (AMPs) play an important role in limiting pathogen entry and colonization. Despite intensive research into the regulation and biochemical properties of AMPs, their exact significance in vivo has remained uncertain due to the challenges of mutating small genes. Fortunately, recent technologies have enabled the mutation of individual AMP genes, overcome previous obstacles, and opened new avenues for research. In this study, we characterized one novel host-defense peptide, Paillotin (IM18, CG33706 ), using loss-of-function mutants. Paillotin is an ancient host defense peptide of Diptera, regulated by the Imd pathway. Loss of Paillotin does not impact the activity of either the Imd or Toll pathways. Importantly, we found that Paillotin mutants are viable but exhibit increased susceptibility to specific infections, particularly Providencia burhodogranariea . Paillotin was further found to contribute synergistically to defense against P. burhodogranariea when combined with other AMPs. However, we did not detect direct microbicidal activity of Paillotin in vitro in our hands. Taken together, our findings identify Paillotin as a novel host defense peptide acting downstream of Imd signaling, advancing our understanding of the Drosophila antimicrobial response.

The nuclear pore complex (NPC) is a large multi-protein structure that enables movement of macromolecules, such as mRNA and proteins, between the nucleoplasm and cytoplasm. There has been great interest in how the physical state of the NPC can influence nuclear-cytoplasmic transport. The hypothesis that the NPC may be mechanosensitive is supported by prior reports showing that the diameter of the NPC increases with nuclear envelope stretch as well as increased ECM stiffness. We therefore sought to develop a biosensor-based approach to determine if the NPC experiences mechanical tension. Using a previously developed FRET-force biosensor, known as TSmod, we developed a gp210 tension sensor. gp210 is a transmembrane nucleoporin, which may serve to anchor the NPC into the nuclear envelope. Using a CRISPR knock-in strategy, we developed a HeLa cell line which expresses the gp210 tension sensor at endogenous levels. Using this sensor, we observed that gp210 forces increase in response to osmotically induced nuclear swelling. Cell attachment, ECM stiffness, the nuclear LINC complex, chromatin condensation, and actomyosin contractility were all observed to influence gp210 forces. Surprisingly, gp210 forces were increased with chromatin relaxation and myosin light chain kinase inhibition, indicating that NPC forces may be differentially regulated from forces on the LINC complex. Our data support a hypothesis where nuclear strain, rather than cytoskeletal forces, is the predominant source for NPC forces. Our studies demonstrate that NPC proteins do experience mechanical tension. We anticipate that the gp210 force sensor will be of use for future studies of NPC mechanobiology.

CRISPR gene activation (CRISPRa) tools have shown great promise for bacterial strain engineering but often require customization for each intended application. Our goal is to create generalizable CRISPRa tools that can overcome previous limitations of gene activation in bacteria. In eukaryotic cells, multiple activators can be combined for synergistic gene activation. To identify potential effectors for synergistic activation in bacteria, we systematically characterized bacterial activator proteins with a set of engineered synthetic promoters. We found that optimal target sites for different activators could vary by up to 200 bases in the region upstream of the transcription start site (TSS). These optimal target sites qualitatively matched previous reports for each activator, but the precise targeting rules varied between different promoters. By characterizing targeting rules in the same promoter context, we were able to test activator combinations with each effector positioned at its optimal target site. We did not find any activator combinations that produced synergistic activation, and we found that many combinations were antagonistic. This systematic investigation highlights fundamental mechanistic differences between bacterial and eukaryotic transcriptional activation systems, and suggests that alternative strategies will be necessary for strong bacterial gene activation at arbitrary endogenous targets.

Background Social behaviour encompasses the wide range of interactions that occur between members of the same species. In humans, disruptions in social behaviour are characteristic of many neuropsychiatric disorders, where both genetic risk factors and synaptic dysfunctions can contribute to the phenotype. Among the genes implicated in synaptic regulation, the synaptic adhesion protein leucine-rich repeat transmembrane protein 4 (LRRTM4) has been identified as a key player in maintaining synaptic function and neuronal circuit integrity. Despite its established role in the nervous system, the potential involvement of LRRTM4 in modulating social behaviour and its contribution to social deficits has yet to be explored. Methods In the current study, we used zebrafish to study how genetic deletion of lrrtm4l1 , a zebrafish orthologue of LRRTM4 , affects sociality. For this, the social behaviour of homozygous lrrtm4l1 knockout (KO) zebrafish was analysed in multiple behavioural assays and the brain transcriptome of mutant animals was investigated by RNAseq. Results KO zebrafish displayed a pro-social phenotype in multiple behavioural assays. Groups of lrrtm4l1 KO zebrafish formed more cohesive shoals and KO individuals spent more time in the vicinity of conspecifics during a social interaction test. They were also less aggressive and in contrast to wild-type zebrafish did not differentiate in their interactions with known and unknown groups of fish. Neurotranscriptomic analysis revealed 560 differentially expressed genes including changes in glutamatergic neurotransmitter signalling, tryptophan- kynurenine metabolism and synaptic plasticity. Conclusion These findings suggest that lrrtm4l1 is an important regulator of social behaviour in zebrafish. In a translational perspective, LRRTM4 is a promising potential therapeutic target that warrants further investigation in the framework of neuropsychiatric conditions characterized by major social impairments.

Barrier epithelia are shielded from the external environment by their apical extracellular matrices (aECMs). The molecular complexity of aECMs has challenged understanding of their organization in vivo . To define the molecular architecture of a model aECM we generated a toolkit of 101 fluorescently tagged aECM components using gene editing in C. elegans , focusing on proteins secreted by the epidermis to form the collagen-rich cuticle. We developed efficient pipelines for modular protein tagging and rapid fluorophore swapping. Most tagged collagens were functional and exhibited exquisitely specific patterning across stages, cell types, and matrix substructures. We define multiple reference markers for key substructures including the little-understood cortical layer, as well as the helical crossed fiber arrays that function as a hydrostatic skeleton to maintain organismal shape. We further tagged >30 members of key aECM protein classes including proteases, protease inhibitors, and lipid transporters. Our standardized markers will allow dissection of the mechanistic basis of aECM spatiotemporal patterning in vivo . Highlights First large-scale protein tagging resource for the apical extracellular matrix Optimization of CRISPR methods for protein tagging including color swaps Tagged proteins are functional and exhibit a high degree of stage-, cell- and compartment specificity Reference localization patterns for multiple aECM compartments and markers for newly defined compartments

Mitochondria play critical roles in energy production and cellular metabolism. Despite the Warburg effect, mitochondria are crucial for the survival and proliferation of cancer cells. Heat Shock Factor 1 (HSF1), a key transcription factor in the cellular heat shock response, promotes malignancy and metastasis when aberrantly activated. To understand the multifaceted roles of HSF1 in cancer, we performed a genome-wide CRISPR screen to identify epistatic interactors of HSF1 in cancer cell proliferation. The verified interactors of HSF1 include those involved in DNA replication and repair, transcriptional and post-transcriptional gene expression, and mitochondrial functions. Specifically, we found that HSF1 promotes cell proliferation, mitochondrial biogenesis, respiration, and ATP production in a manner dependent on TIMM17A, a subunit of the inner membrane translocase. HSF1 upregulates the steady-state level of the short-lived TIMM17A protein via its direct target genes, HSPD1 and HSPE1, which encode subunits of the mitochondrial chaperonin complex and are responsible for protein refolding once imported into the matrix. The HSF1- HSPD1/HSPE1-TIMM17A axis remodels the mitochondrial proteome to promote mitochondrial translation and energy production, thereby supporting robust cell proliferation. Our work reveals a mechanism by which mitochondria adjust protein uptake according to the folding capacity in the matrix by altering TIM complex composition.

The amyloid precursor protein ( APP ) is processed by multiple enzymes to generate biologically active peptides, including amyloid-β (Aβ), which aggregates to form the hallmark pathology of Alzheimer’s disease (AD). Aβ is produced through an initial β-secretase cleavage of APP, generating a 99-amino acid C-terminal fragment (APP-C99). Subsequent cleavage of APP-C99 by γ-secretase produces Aβ peptides of varying lengths. To better understand the transcriptional regulation of Aβ production, we employed long-read RNA sequencing and identified previously unannotated transcripts encoding APP-C99 with an additional methionine residue (APP-C100), generated independently of β-secretase cleavage. These transcripts are expressed separately from full-length APP , and we observed that cells lacking full-length APP can still produce Aβ through these shorter isoforms. Importantly, mass spectrometry analysis of cerebrospinal fluid (CSF) revealed peptides consistent with the methionine-extended Aβ species, supporting the in vivo translation of these transcripts. Our findings reveal an alternative pathway for Aβ generation and aggregation, highlighting a potential new target for modulating Aβ accumulation in AD.

Comparative anatomical studies of primates and extinct hominins, including Neanderthals, show that the modern human brain is characterised by a disproportionately enlarged neocortex relative to the striatum. To explore the molecular basis of this difference, we screened for missense mutations that are unique to modern humans and occur at high frequency and that alter post-translational sites. One such mutation was identified in DCHS1 , a protocadherin family gene, and it was found to disrupt an N-glycosylation site in modern humans. Using CRISPR/Cas9-editing we introduced into human-induced pluripotent stem cells (hiPSCs) this ancestral DCHS1 variant present in Neanderthals and other primates, representing the ancestral state before the modern human-specific substitution. Leveraging hiPSCs-derived neural organoids, we observed an expansion of striatal progenitors at the expense of the neocortex, mirroring the anatomical distribution seen in non-human primates. We further identify the ephrin receptor EPHA4 as a binding partner of DCHS1 and show that modern human-specific alterations in DCHS1 modulate EPHA4-ephrin signalling, contributing to a gradual shift in the neocortex-to-striatum ratio - a hallmark of brain organisation in our species.

The diploid genome of the fungal pathogen Candida albicans is highly heterozygous, with most allele pairs diverging at either the coding or regulatory level. When faced with selection pressure like antifungal exposure, this hidden genetic diversity can provide a reservoir of adaptive mutations through loss of heterozygosity (LOH) events. Validating the potential phenotypic impact of LOH events observed in clinical or experimentally evolved strains can be difficult due to the challenge of precisely targeting one allele over the other. Here, we show that a CRISPR-Cas9 system can be used to overcome this challenge. By designing allele-specific guide RNA sequences, we can induce targeted, directed LOH events, which we validate by whole-genome long-read sequencing. Using this approach, we efficiently recapitulate a recently described LOH event that increases resistance to the antifungal fluconazole. Additionally, we find that the recombination tracts of these induced LOH events have similar lengths to those observed naturally. To facilitate future use of this method, we provide a database of allele-specific sgRNA sequences for Cas9 that provide near genome-wide coverage of heterozygous sites through either direct or indirect targeting. This approach will be useful in probing the adaptive role of LOH events in this important human pathogen.

Malaria merozoite surface proteins (MSPs), are thought to have important roles in red blood cell (RBC) invasion and their exposure on the parasite surface makes them attractive vaccine candidates. However, their role in invasion has not been directly demonstrated and their biological functions are unknown. One of the most abundant proteins is Pf MSP2, which is likely an ancestral protein that has been maintained in the Plasmodium falciparum lineage and is a focus of vaccine development, whose function remains unknown. Using CRISPR-Cas9 gene-editing, we removed Pf MSP2 from two different P. falciparum lines with no impact on parasite replication or phenotype in vitro , demonstrating that it is not essential for RBC invasion. However, loss of Pf MSP2 led to increased inhibitory potency of antibodies targeting other merozoite proteins involved in invasion, particularly Pf AMA1. In a solid-phase model, increasing concentrations of Pf MSP2 protein reduced binding of different antibodies against Pf AMA1 in a dose dependent manner. These data suggest that Pf MSP2 can modulate the susceptibility of merozoites to protective inhibitory antibodies. The results of this study change our understanding of the potential functions of Pf MSP2 and establishes a new concept in malaria where a surface protein can reduce the protective efficacy of antibodies targeting a different antigen. These findings have important implications for understanding malaria immunity and informing vaccine development.

Abstract In sustainable aquaculture, probiotics offer a promising alternative to antibiotics for improving shrimp health. Bacillus sp. KNSH11, isolated from the intestine of whiteleg shrimp ( Litopenaeus vannamei ), was characterized to evaluate its probiotic potential. The strain, a Gram-positive, rod-shaped bacillus, exhibited exceptional spore formation efficiency (> 99%), ensuring resilience in challenging environments. Functional assays demonstrated that KNSH11 maintained high viability at pH 2–4, in the presence of bile salts, at temperature up to 95°C, and under lysozyme exposure, indicating tolerance to gastrointestinal and processing stresses. Metabolic profiling indicated significant lactic acid production with minimal acetate and propionate, distinguishing it from conventional lactic acid bacteria. KNSH11 also displayed strong antioxidative activities and moderate antibiofilm effects against pathogens. Antibiotic susceptibility testing revealed sensitivity to amoxicillin (30 µg/disc), chloramphenicol (30 µg/disc), kanamycin (30 µg/disc) and tetracyclines (30 µg/disc), but resistance to ampicillin (10 µg/disc) and penicillin (10 µg/disc). Whole genome sequencing (WGS) confirmed the absence of virulence factors and identified mobile genetic elements, a CRISPR/Cas system, and gene clusters potentially encoding bacteriocins. Collectively, these findings suggest that Bacillus sp. KNSH11 is safe, eco-friendly probiotic with significant potential to enhance shrimp health and advance sustainable aquaculture.

Abstract Natural CRISPR-Cas9 systems provide a rich resource for developing genome editing tools with diverse properties, including genome size, protospacer preference, and PAM specificity. In this study, we screened a panel of 11 Cas9 nucleases orthologous to CjCas9 using a GFP activation assay and identified seven active nucleases. Among these, Cj4Cas9 emerges as particularly noteworthy due to its compact genome size (985 amino acids) and unique PAM preference (5’-NNNGRY-3’). Cj4Cas9 demonstrates efficient disruption of the Tyr gene in mouse zygotes, resulting in an albino phenotype. Furthermore, when delivered via AAV8, Cj4Cas9 achieves efficient genome editing of the Pcsk9 gene in mouse liver, leading to reduced serum cholesterol and LDL-C levels. To enhance its utility, we engineered Cj4Cas9 for higher activity by introducing L58Y/D900K mutations, resulting in a variant termed enCj4Cas9. This variant exhibits a two-fold increase in nuclease activity compared to the wild-type Cj4Cas9 and recognizes a simplified N3GG PAM, considerably expanding its targeting scope. These findings highlight the potential of Cj4Cas9 and its high-activity variants for both fundamental research and therapeutic applications.

Abstract CBFA2T3::GLIS2 fusion positive pediatric acute myeloid leukemia (AML) remains one of the worst prognostic AML subgroups. To uncover innovative targeted therapeutic approaches in this disease subtype we performed genome-scale CRISPR-Cas9 screening that highlighted a strong, selective dependency on JAK2 compared to other types of cancer. Using a doxycycline-inducible JAK2 knockout (KO) system, we validated JAK2 dependency in CBFA2T3::GLIS2 cell lines, observing impaired proliferation in vitro and in vivo and induced apoptosis with JAK2 KO. Both type I (ruxolitinib) and type II (CHZ868) JAK2 inhibitors showed selective in vitro activity in CBFA2T3::GLIS2 positive AML models. To identify resistance and sensitizer mechanisms to JAK2 inhibitors, we used CRISPR-Cas9 ruxolitinib anchor screening in CBFA2T3::GLIS2 AML. sgRNAs targeting negative regulators of the MAPK pathway were enriched in the ruxolitinib-treated cells. Similarly, CBFA2T3::GLIS2 AML sublines grown to resistance under chronic ruxolitinib treatment expressed pathogenic NRAS mutations. Both approaches converged on MAPK pathway activation as a resistance mechanism to ruxolitinib treatment. Combining ruxolitinib with MEK inhibitors showed a synergistic effect in cell lines and patient-derived xenograft (PDX) cells expressing the fusion and in vivo activity in a CBFA2T3::GLIS2 AML PDX, suggesting a potential approach to target this signaling circuitry in this poor outcome AML subtype.

Abstract Background Enhanced metabolic and mitochondrial activity inherent in actively proliferating cancer cells is associated with intracellular redox imbalance that impacts cellular viability. To restore redox homeostasis cancer cells evolve to activate redox protective mechanisms. This differential activation of redox defense pathways compared to normal cells provides a therapeutic window for novel targeted therapies in cancer. Although the heme metabolism emerges as a crucial regulator of redox homeostasis and iron metabolism in cancer cells with frequent alteration in breast cancer, it remains largely unexplored, and no targeted translational approaches have been developed. Heme-regulated redox homeostasis is coordinately maintained through biosynthetic and degradation pathways. As a byproduct of TCA cycle, cytotoxic heme is initially derivatized by heme oxygenases and progressively metabolized to the potent antioxidant bilirubin by two non-redundant biliverdin reductases, BLVRA and BLVRB. BLVRB overexpression has been observed in breast cancers, although its function in breast cancer pathogenesis remains unknown. Methods CRISPR/Cas9 deletion of BLVRB in multiple breast cancer cell lines demonstrated its profound effect on intracellular redox state and cell proliferation in vitro and xenograft models. Integrated proteomic, metabolomic, and lipidomic studies identified and validated BLVRB–mediated adaptive metabolic responses required for breast cancer cell cytoprotection. Results We have established BLVRB as a requisite component of the pro-survival redox defense mechanism in breast cancer cells. Targeted deletion of BLVRB induces reductive stress, leading to alterations in endoplasmic reticulum proteostasis and lipid composition. These defects impact plasma membrane functionality and endosomal recycling of multiple oncogenic receptors, such as HER2 and transferrin receptors. Conclusions These data collectively identify BLVRB as a novel metabolic target in breast cancer, distinct from other redox-regulating pathways. This study, along with our recent progress in developing novel specific BLVRB inhibitors, offers a unique translational opportunity for targeted therapies in personalized breast cancer medicine.

Abstract The CRISPR/Cas9 based technology has been used for sequential gene editing in E. coli . The plasmids carrying the sgRNA and/or Cas9 genes need to be cured after each round of editing. Curing of these plasmids, particularly the sgRNA plasmid, limits the efficiency of sequential gene editing. In this study, a lethal endotoxin ( ccd B) based counter-selection was established for improving the overall efficiency of sequential gene editing in E. coli . This approach was validated for sequential editing (deletion) of cst A and pps A genes. The experimental results showed that the transformation efficiency sgRNA plasmid (pTargetF- tcr -P L - ccd B-N20) reached to 10 8 -10 9 cfu / µg -DNA , resulting in a 90% of recombination rate for the target gene ( cst A and pps A). Upon completion of cst A gene editing, the sgRNA plasmid (pTargetF- tcr -P L - ccd B-N20( cst A)) were effectively cured through ccd B based counterselection at 42°C, with a 43.75% efficiency. At the end of sequential editing of pps A gene, both Cas9 (25A) and sgRNA (pTargetF- tcr -P L - ccd B-N20( pps A)) plasmids were cured simultaneously through the sac B and ccd B based counterselections by incubating the cells on LB-sucrose (5%) plate at 42°C, achieving a curing rate of 100% for Cas9 plasmid (25A), and 37.5% for sgRNA plasmid (pTargetF- tcr -P L - ccd B-N20( pps A)). These results demonstrated that the endotoxin ( ccd B) based counterselection improved the transformation efficiency of sgRNA plasmid, the recombination rate of the editing target gene, the curing rate of sgRNA plasmid, and the overall efficiency of sequential gene editing.

ABSTRACT Protein translation regulation is critical for cellular responses and development, yet how disruptions during the elongation stage shape these processes remains incompletely understood. Here, we identify and validate a single amino acid substitution (P55Q) in the ribosomal protein RPL-36A of Caenorhabditis elegans that confers complete resistance to high concentrations of the elongation inhibitor cycloheximide (CHX). Heterozygous animals carrying both wild-type RPL-36A and RPL-36A(P55Q) exhibit normal development but intermediate CHX resistance, indicating a partial dominant effect. Leveraging RPL-36A(P55Q) as a single-copy positive selection marker for CRISPR-based genome editing, we introduced targeted modifications into multiple ribosomal protein genes, confirming its broad utility for altering essential loci. In L4-stage heterozygotes, where CHX-sensitive and CHX-resistant ribosomes coexist, ribosome profiling revealed increased start-codon occupancy, suggesting early stalling of CHX sensitive ribosomes. Chronic CHX reduced ribosome collisions, evidenced by fewer disomes and unchanged codon distributions in monosomes. Surprisingly, prolonged elongation inhibition did not activate well characterized stress pathways–including ribosome quality control (RQC), the ribotoxic stress response (RSR), or the integrated stress response (ISR)–as indicated by absence of changes in RPS-10 ubiquitination, eIF2α phosphorylation, PMK-1 phosphorylation, or the transcriptional upregulation of ATF-4 target genes. Instead, RNA-normalized ribosome footprints revealed gene-specific changes in translation efficiency, with nucleolar and P granule components significantly decreased while oocyte development genes were increased. Consistent with these observations, we detected premature oogenesis in L4 animals, suggesting that partial translation elongation inhibition reshapes translation efficiency, to fine-tune developmental timing.

Over the last two decades, new in vivo and in cellulo imaging technologies have uncovered the inherently dynamic nature of transcriptional regulation in embryonic development and, in particular, in the fruit fly D. melanogaster .These technologies have made it possible to characterize the subnuclear and single-molecule dynamics of transcription factors. However, a lack of appropriate fluorescent protein fusions has, until now, limited these studies to only a few of the dozens of important transcription factors in the fruit fly gene regulatory network dictating early development. Here, we report the creation of four new fluorescent protein fusions to Dorsal, a member of the NF-κB/Rel family that initiates dorsal-ventral patterning. We generated and characterized two bright fluorescent protein fusions for Dorsal, meGFP and mNeonGreen, and two photoconvertible fluorescent protein fusions, mEos4a and Dendra2. We show that removal of the DsRed2 cassette commonly used to mark the CRISPR integration restores endogenous Dorsal mRNA and protein levels and enables the fusion allele to rescue a dorsal null allele, meeting the gold standard for endogenous function of a tagged protein in a fruit fly. We then demonstrate that our bright fluorescent protein fusions can be used to dissect the spatiotemporal dynamics of stable Dorsal clusters that traverse the nucleoplasm and uncovered that these clusters preferentially interact with active sites of Dorsal-modulated transcription. We further demonstrate that our photoconvertible fluorescent protein fusions make it possible to detect individual molecules of Dorsal in the nuclei of developing embryos. These new fluorescent protein fusions constitute a valuable resource for the community to elucidate the role of Dorsal activator dynamics in dictating fruit fly early embryonic development.

Abstract The prognosis of colorectal cancer (CRC) is often fatal, and the underlying mechanisms are unclear. Dysregulation of enhancers is involved in several tumors, and this area is expected to become a new frontier in tumor therapy. Here, we identified a novel activated enhancer ‘Enhancer X’ from the Gene Expression Omnibus database and experimentally verified its associated RNA (EX-eRNA) in CRC. To date, there are no reports on the function of Enhancer X, and its role in CRC remains unknown. We aimed to investigate the biological functions and underlying mechanism of Enhancer X in CRC. EX-eRNA expression was elevated in CRC cells and tissues and positively correlated with CRC metastasis and shorter survival. Enhancer X knockout (CRISPR/Cas9) or EX-eRNA knockdown (siRNAs) inhibited CRC cell growth in vivo and in vitro. Mechanistically, Enhancer X activation and transcription to EX-eRNA require the binding of CEBPB to a “GTTGTGTCAC” motif within the enhancer region. Subsequently, EX-eRNA, serves as a scaffold for MED25 to stabilize the chromatin loop between Enhancer X region and the KRT80 promoter, thereby promoting KRT80 transcription and driving CRC tumorigenesis. Thus, we conclude that novel enhancer X activated by CEBPB drives colorectal carcinogenesis via EX-eRNA-mediated DNA loop promotion of KRT80 transcription, and that targeting the CEBPB / Enhancer X / EX-eRNA / KRT80 molecular axis constitutes a promising approach to treat CRC.

Pyridoxine-dependent epilepsy (PDE) is a rare neurometabolic disorder of lysine catabolism caused by bi-allelic variants in ALDH7A1 . This enzyme deficiency leads to the accumulation of neurotoxic metabolites, pyridoxal-phosphate inactivation and consequently severe neurological symptoms. Current treatments, including vitamin B6 supplementation and lysine-restricted diets, partially alleviate seizures and intellectual disability but are not curative. To explore underlying mechanisms and potential therapies, we generated patient-derived human induced pluripotent stem lines (hiPSC) that were subsequently differentiated into astrocytes, the primary source of ALDH7A1 in the brain and key regulators of metabolic homeostasis. Metabolomic analyses confirmed elevated PDE biomarkers, and RNA sequencing revealed gene expression changes consistent with increased oxidative stress. Oxidative damage was validated by markers of DNA oxidation and lipid peroxidation. In addition, dysregulated oxygen consumption rates suggested mitochondrial dysfunction in PDE astrocytes. Notably, these pathological phenotypes were alleviated by downregulating AASS , the first enzyme of the lysine catabolism, by using CRISPR/Cas9 editing or antisense oligonucleotides (AON). This demonstrates that lysine catabolism underlies these phenotypes and highlights the therapeutic potential of AON therapy targeting AASS to reduce neurotoxic metabolite accumulation. These findings provide a promising strategy for developing targeted treatments for PDE and other rare neurometabolic disorders.

Abstract Background: Genomic editing technologies provide rich opportunities to approach inherited human diseases, but gene delivery strategies are still challenging. While engineered induced pluripotent stem cells or adeno-associated viral vectors may be associated with severe adverse effects, mesenchymal stromal cells (MSC) have abundantly been used for various cell therapeutic strategies and have been proven to be safe in a large number of clinical trials. MSC can be expanded ex vivo on a large scale, and genetic engineering of these somatic stem cells may therefore offer an alternative mode of gene delivery. Methods: We applied the guide RNA-directed CRISPR/Cas9 synergistic activation mediator (SAM) technology in immortalized (iMSC) and primary human MSC (pMSC) in order to activate gene expression of CEBPA and CIITA . Results: After meticulous protocol optimization, both genes were successfully induced in MSC. For CIITA induction, we confirmed expression of the downstream target HLA-DR on mRNA and protein level. Unlike adipogenic, chondrogenic or osteogenic genes, which are naturally induced in MSC upon in vitro differentiation, HLA class II molecules are not constitutively expressed in MSC. Lentiviral and transposon-based delivery strategies were applied, but only with lentiviral transduction of the SAM sequences, CEBPA, CIITA, HLA-DR gene and HLA-DR protein expression were achieved in pMSC. Conclusion: Lentiviral transduction of the guided SAM system was capable to induce transcriptionally silent genes in iMSC and pMSC. The resulting genetically engineered somatic stem cells may be suitable and promising for cell-based therapeutic strategies.

Abstract Background Alzheimer’s disease (AD) is characterised by progressive cognitive decline and accumulation of pathological markers such as β-amyloid (Aβ) plaques and Tau tangles. Emerging evidence suggests these markers can also be detected in the retina, positioning it as a potential surrogate for investigating AD pathophysiology. The retinal pigment epithelium (RPE) shares features with the brain and is critical for retinal health, yet its role in AD pathology remains underexplored. Methods We generated RPE cells from human induced pluripotent stem cells carrying the PSEN1 H163R pathogenic variant for AD, alongside its CRISPR-corrected isogenic control. AD-associated phenotypes were assessed. Results RPE cell cultures from the two cohorts displayed expression of Aβ and Tau, with notable differences in levels and organisation. Total Aβ 1−42 and Aβ 1−42:1−40 ratio in PSEN1 H163R RPE cell lysates were significantly elevated compared to the CRISPR isogenic controls and volume of Aβ + deposits was significantly larger in PSEN1 H163R RPE cells. Total and phosphorylated Tau proteins were also detected in both cohorts, with altered spatial organisation and localisation of pTau in PSEN1 H163R . Proteomic profiling identified more than 1,800 significantly dysregulated proteins in PSEN1 H163R RPE cells, including key AD-related proteins such as MAPT, APP, APBB1 and NRBF2. Upregulated pathways involved autophagy, intracellular trafficking and neurodegeneration, while downregulated pathways implicated mitochondrial respiration, RNA metabolism, and protein folding. Proteomics analysis of conditioned media further revealed altered secretion of matrix-associated proteins as well as increased APOE and APP in PSEN1 H163R RPE samples. PSEN1 H163R RPE cells demonstrated dysregulation in melanosome biogenesis, marked by decreased expression of core melanogenic proteins (PMEL, TYR, DCT) by proteomics analysis; and altered melanosome morphology and pigmentation by electron microscopy. Conclusion In conclusion, these findings support the RPE as a relevant and accessible in vitro model for AD research, offering insights into the role of PSEN1 in Aβ and Tau dysregulation, disease mechanisms and melanosome biogenesis, providing a promising approach to understand PSEN1 biology in the context of disease and potential biomarker discovery. It is also the first to describe a relationship between PSEN1 H163R and melanosomes in a human cellular model.

The rapid development of CRISPR-Cas gene editing technologies has revolutionized genetic medicine, offering unprecedented precision and potential for treating a wide array of genetic disorders. However, assessing the risks of unintended gene editing effects remains critical, and is complicated by new editing modalities and unclear analytical guidelines. We present UNCOVERseq (Unbiased Nomination of CRISPR Off-target Variants using Enhanced RhPCR), an improved in cellulo off-target nomination workflow designed to sensitively nominate off-target sites (<0.01% editing) with defined input requirements and analytical process controls to provide empirical performance evidence across diverse circumstances. Using this workflow, we nominated off-targets across 192 guide RNAs (gRNAs) and demonstrated superior performance compared to existing methodologies. We identified a subset of six gRNAs with a dynamic range of specificity and confirmed the relevance and high true positive rate of our nomination method, providing relative risk assessments for multiple modalities ( S.p. Cas9 and derived high-fidelity variants / base editors) in a translational system involving hematopoietic stem and progenitor cells (HSPCs). Additionally, we established that double-strand break (DSB) editing retains a strong, positive rank correlation to single-strand break (SSB)-mediated base editing, highlighting the importance of DSB nomination sites as candidate loci for base editing. Overall, UNCOVERseq improves informed risk assessment of gene editing in translational systems by enhancing the quality of off-target nomination.

ABSTRACT We demonstrate here the use of optical genome mapping (OGM) to detect genetic alterations arising from gene editing by various technologies in human induced pluripotent stem cells (iPSCs). OGM enables an unbiased and comprehensive analysis of the entire genome, allowing the detection of genomic structural variants (SVs) of all classes with a quantitative variant allele frequency (VAF) sensitivity of 5%. In this pilot study, we conducted a comparative dual analysis between the parental iPSCs and the derived cells that had undergone gene editing using various techniques, including transposons, lentiviral transduction, and CRISPR-Cas9-mediated safe harbor locus insertion at the adeno-associated virus integration site 1 (AAVS1). These analyses demonstrated that iPSCs that had been edited using transposons or lentiviral transduction resulted in a high number of transgene insertions in the genome. In contrast, CRISPR-Cas9 technology resulted in a more precise and limited transgene insertion, with only a single target sequence observed at the intended locus. These studies demonstrate the value of OGM to detect genetic alterations in engineered cell products and suggests that OGM, together with DNA sequencing, could be a valuable tool when evaluating genetically modified iPSCs for research and therapeutic purposes.

Gene editing technologies have opened the possibility of directly targeting viral DNA in therapeutic applications. In chronically infected hepatocytes with hepatitis B virus (HBV), covalently closed circular DNA (cccDNA) serves as the master template for viral transcripts and gene products. In the present study, we evaluated the outcomes of anti-HBV multiplex gene editing with the CRISPR-Cas9 endonuclease from Staphylococcus aureus (SaCas9) using primary human hepatocytes (PHHs) and HBV mouse models. Nonviral delivery of SaCas9-encoding mRNA and a pair of HBV-targeting guide RNAs (gRNAs) substantially reduced viral biomarkers and intrahepatic HBV DNA copies in vitro and in vivo, suggesting that fragmentation of HBV DNA primarily leads to its degradation. Hybridization capture sequencing analyses indicated that small insertions and deletions (indels) and structural variants including excisions and inversions of the viral sequences were accumulated in the residual HBV DNA. These assays also demonstrated that transient expression of the HBV-targeting SaCas9 significantly suppressed random integration of HBV DNA, while this therapeutic approach was unlikely to affect chromosomal translocations involving viral copies. Taken together, our results suggest that anti-HBV multiplex gene editing eliminates viral DNA from chronically infected hepatocytes, potentially reducing the risk of hepatocarcinogenesis associated with HBV DNA integration.