Differential gene expression establishes the distinct physiology and morphology of cell types in an animal body. Single-cell sequencing and volume EM represent milestones toward the characterization of cell types, yet are difficult to combine for a comprehensive view on the cellular genotype-phenotype link. Here, we map a whole-body single-cell transcriptome into the PlatyBrowser, a multimodal cellular atlas for the marine annelid Platynereis dumerilii , and establish this combination uniquely for an entire animal. We learn that, in the 6-days-old worm, the majority of genes are tightly co-regulated to jointly implement one of eight major cellular morphotypes representing epidermis, gut, vasculature, myofibres, glia, motile cilia, glands, or neurons. Focusing on neurons, we uncover 14 families that by transcription factor identity, axonal projection, or sensory-secretory apparatus resemble conserved neuron types found in vertebrates, insects, or nematodes. We hypothesize that these existed in urbilaterian ancestors and represent the ancient core of nervous system centralization.

Background Macrophages adopt activation states along a spectrum from pro- to anti-inflammatory, enabling appropriate responses to pathogens and environmental cues. Dysregulated inflammatory macrophage activation contributes to diseases including sepsis, rheumatoid arthritis, cancer, and atherosclerosis. Epigenetic processes such as DNA methylation and histone modification prime macrophages for activation, and several histone modifying enzymes (HMEs) have been implicated in this regulation. Objective To systematically identify histone modifying enzymes that regulate inflammatory macrophage activation. Methods We performed a CRISPR knockout screen with single-cell RNA-seq readout (CROP-seq) targeting 92 macrophage-expressed HMEs in immortalized LPS-activated mouse bone marrow-derived macrophages (BMDMs). The resulting single-cell transcriptomes were analyzed to identify significant perturbations. Kdm5c was selected for experimental validation in mouse BMDMs, and its expression pattern was compared with macrophage subsets from human atherosclerotic plaques using scRNA-seq data. Results The CROP-seq screen identified Prmt6, Carm1, Kat2b , and Kdm5c as top regulators of inflammatory macrophage activation. Validation in a KO cell line revealed loss of Kdm5c suppressed inflammatory tone at baseline but led to an exaggerated transcriptional response to LPS stimulation, indicating a role for Kdm5c in balancing tonic and inducible activation. A weighted gene module derived from Kdm5c -deficient macrophages was enriched in inflammatory macrophages in human atherosclerotic plaques. Conclusion Our findings demonstrate the value of CROP-seq screening to dissect the epigenetic control of macrophage activation. We also identify Kdm5c-mediated histone demethylation as a key mechanism modulating inflammatory macrophage activation.

NUT carcinoma (NC) is an aggressive malignancy driven by NUTM1 gene rearrangements with limited therapeutic options. Here, we show that direct suppression of NUTM1 using CRISPR/Cas9 induces squamous-like differentiation and upregulates TROP2 expression in NC cells. Building on this finding, we developed a TROP2–interferon beta (IFN-β) mutein immunocytokine that selectively targets TROP2-expressing tumors. Combined NUTM1 suppression and TROP2-targeted immunotherapy synergistically enhanced cytotoxic and immune-mediated responses in vitro . Transcriptomic and spatial analyses of NC patient tumors revealed that differentiation status correlates with TROP2 expression, upregulated immune pathways, and favorable clinical outcomes. Our results suggest that overcoming differentiation blockade not only alters tumor phenotype but also creates a more immune-permissive microenvironment. These findings highlight the therapeutic potential of sequential tumor reprogramming followed by targeted immunotherapy in treating NC and propose a broader strategy for overcoming differentiation blockade in fusion-driven cancers.

Acute myeloid leukemia (AML) is a heterogeneous disease that arises from dysregulated myeloid proliferation. We performed a high-throughput CRISPR interference (CRISPRi) screen in the THP-1 monocytic cancer cell line to identify long noncoding RNAs (lncRNAs) that play a role in contributing to cell proliferation. Our screen identified INSTAR (Intergenic Nuclear Suppressor lncRNA Targeting Adjacent Regulator SFMBT2 ) as a top candidate. RNA-seq on INSTAR deficient THP-1 cells revealed transcriptional changes in genes involved in cell proliferation as well as other cellular processes. Loss of INSTAR selectively reduced expression of its neighboring gene, SFMBT2 . Functional assays confirmed that both genes suppress cell growth, revealing a cis-regulatory mechanism in which INSTAR regulates SFMBT2 expression to control monocyte proliferation. Here, we leverage high-throughput screening to rapidly pinpoint functional lncRNAs providing novel insights into a key regulatory locus consisting of INSTAR and SFMBT2 which could be critical for better understanding dysregulation contributing to acute myeloid leukemia.

Macroautophagy/autophagy is an essential developmental and homeostatic process, defined by the endolysosomal degradation of intracellular components and pathogens. Dysfunctional autophagy is implicated in complex human disease, yet reports of congenital autophagy disorders were considered exceedingly rare until the recent report of several unrelated families with bi-allelic variants in ATG7 , encoding a core autophagy effector complementing the report of two individuals harbouring ATG5 variants. We now report six affected individuals from five families with bi-allelic ATG12 variants with complex neurological phenotypes overlapping those of ATG5 and ATG7 patients, including developmental delay, congenital ataxia and cerebellar hypoplasia. Structural modelling implicated a potential disruption of the important ATG12–ATG5-ATG16N-ATG3 complex. Biochemical analyses of patient-derived primary fibroblasts from members of two affected families confirmed the loss of stable ATG12–ATG5 conjugate in one family and altered autophagic flux in one unrelated family. HeLa cell models demonstrate a decrease in ATG12–ATG5 conjugate and reduced autophagic flux in response to starvation using the Halo Tag processing assay. Yeast complementation studies demonstrated that equivalent missense atg12 variants were unable to fully recover the biochemical defect in atg12 null yeast and also demonstrated a reduced delivery of autophagy substrates to the yeast’s degradative compartment. Functional studies in zebrafish models demonstrated global developmental delay, impaired brain function, and pre-adulthood lethality. Our findings underscore the pivotal role of efficient autophagy in maintaining human neural integrity and emphasise the importance of this emerging group of congenital autophagy disorders, thereby expanding our understanding of adaptive homeostasis in human health and disease.

ABSTRACT CRISPR/Cas9-based mosaic analysis is a powerful tool for in vivo genetics but is limited by cytotoxicity and mutagenesis associated with DNA double-strand breaks (DSBs). Here, we establish Cas9-derived nickases as safer and more reliable alternatives for inducing mitotic recombination in Drosophila . We demonstrate that single-strand nicks are sufficient to generate mosaic clones and systematically dissect the parameters governing this process. We find that clone frequency can be controlled by the gRNA nicking pattern, with two distant nicks on the same DNA strand synergistically enhancing recombination by over nine-fold compared to a single nick. Based on these findings, we propose a mechanistic model for nick-induced crossover and provide a versatile toolkit for generating tissue-specific nickases. This work establishes nickase-based MAGIC as a superior method for high-fidelity clonal analysis, enabling more precise investigation of gene function in development and disease. SIGNIFICANCE STATEMENT The CRISPR/Cas9-based mosaic technique, MAGIC, is a versatile tool for in vivo biological investigations. However, its reliance on DNA double-strand breaks (DSBs) can cause significant, unintended cell damage. Here we establish that Cas9-derived nickases, which create gentler single-strand nicks, are a superior alternative. We show that nickases safely induce genetic mosaics in Drosophila by avoiding this cellular toxicity. By systematically dissecting the process, we discovered principles of gRNA design that allow clone frequencies to be ‘tuned’ for different experimental needs. This work provides a new mechanistic model for nick-induced genetic exchange, a high-fidelity “nickase-MAGIC” method, and a versatile toolkit for precision clonal analysis.

CD4⁺ T cell differentiation is orchestrated by coordinated signaling, transcriptional, and epigenomic programs, yet how signaling connects to chromatin and genetic variation in human T cells remains unclear. Here, we generated an integrative multi-omics map of human CD4⁺ T cell activation and differentiation, combining phosphoproteomics, transcriptomics, and chromatin accessibility under Th0, Th1, and iTreg polarization. Within 10 minutes of activation, we observed rapid phosphorylation changes of RNA-binding proteins accompanied by degradation of effector-associated transcripts, preceding chromatin remodeling and later transcriptional activation of the same genes. Moreover, our data highlights how site-specific phosphorylation refines TF activity during T cell differentiation and activation, and identifies CDK1 as a regulator of Th1 effector function. Indeed, we found that a low dose of CKD1 inhibition impairs IFN-γ expression and pro-inflammatory differentiation, while preserving regulatory features in iTregs. Single-cell multi-omic profiling upon CDK1 inhibition revealed how CDK1 activity shapes subset-specific gene regulatory networks, which are enriched for genetic variants associated with immune-traits. Specifically, CDK1-sensitive TFs, including IRF8, connect immune trait heritability to enhancer accessibility at IFNG and TNF loci. Together, these results establish CDK1 as a signaling hub that couples phosphorylation to gene regulation and genetic risk, with therapeutic relevance in autoimmune disease.

ABSTRACT The high mortality associated with tuberculosis (TB), alongside the lack of efficient therapeutics against emerging multidrug-resistant Mycobacterium tuberculosis ( Mtb ) strains, emphasizes the need to identify novel antitubercular targets. Mycobacterial peptidoglycan, displaying characteristic modifications comprising the amidation of D- iso -glutamate (D- i Glu) and the N -glycolylation of muramic acid, is a promising therapeutic target. The genes encoding the enzymes mediating these PG modifications ( murT / gatD and namH ) were silenced in Mtb using CRISPR interference (CRISPRi) to investigate their impact on β-lactam susceptibility and host immune responses. First, qRT-PCR confirmed successful target mRNA knockdown, with variable repression efficiency based on the selected sgRNA, PAM strength, and target site. Phenotypic characterization through spotting dilution and growth curve assays corroborated the essentiality of D- i Glu amidation for mycobacterial survival, in contrast to the N -glycolylation of muramic acid. Moreover, susceptibility assays showed that both PG modifications contribute to β-lactam resistance, with sgRNA2-mediated murT knockdown substantially increasing β-lactam and isoniazid susceptibility. Furthermore, checkerboard assays showed reductions in the minimum fractional inhibitory concentration index (FICI min ) value for AMX/MEM+CLA and EMB combinations following the depletion of both PG modifications, with significant differences observed upon namH knockdown. Additionally, D- i Glu amidation was uncovered as a determinant of Mtb survival within THP-1-derived macrophages at 6 days post-infection. Infection of THP-1-derived macrophages with MurT/GatD-depleted Mtb upregulated IL-1β and downregulated IL-10, whereas NamH depletion caused upregulation of both IL-1β and IL-10. Altogether, our findings unveiled the potential of targeting these PG modifications for the development of innovative therapeutic regimens against TB.

Abstract Genome editing with CRISPR-Cas9 offers a powerful approach for enhancing enzyme production in microorganisms. This study aimed to genetically engineer the lacZ gene in Escherichia coli using CRISPR-Cas9 to evaluate its impact on asparaginase production during submerged fermentation with rice bran serving as a glucose source. Both edited and wild-type E. coli strains were cultured at optimal conditions to produce and characterize asparaginase. The edited E. coli formed distinct colonies, displaying a blue phenotype when exposed to Cas9 without sgRNA or arabinose, yielding a total of 96 colonies. No colonies were observed when Cas9 and sgRNA were present without arabinose, while the addition of Cas9 and arabinose without sgRNA resulted in 309 blue colonies. With Cas9, sgRNA, and arabinose present, repair activation produced 114 distinct white colonies. The editing of the lacZ gene was validated through multiplex PCR and gel electrophoresis, with bands at 650 bp indicated lacZ gene editing, while bands at 1,100 bp indicated the wild-type. Asparaginase production was assessed using plate method assay, submerged fermentation using rice bran as a glucose source, and subsequent purification via ammonium sulfate precipitation and ion-exchange chromatography. Ion-exchange chromatography revealed enhanced purity and activity in the edited strain, with peak activity observed at an elution of 80 mL. The CRISPR-Cas9 edited strain exhibiting significantly higher enzyme activity (1.2 ± 0.002 U/ mL) compared to the wild-type (0.8 ± 0.005 U/mL). Both strains demonstrated maximum asparaginase activity at 40 o C and pH 7. This study concludes that CRISPR-Cas9 meditated lacZ gene editing in E. coli improves its ability to utilize rice bran as a substrate, significantly enhancing asparaginase production. These findings highlight the potential of genetic engineering and agricultural by-products for sustainable enzyme production.

Daily sleep-wake cycle is a conserved behaviour defined by locomotion quiescence and enhanced responsive threshold to sensory stimuli. Both circadian rhythm and a homeostatic process determine the daily sleep profile, which is also regulated by environmental light, a major sensory input to regulate circadian rhythm and alertness. With decades of investigation in Drosophila , the cellular and circuital mechanism underlying light-mediated circadian synchronization are well-established, yet the direct relationship between light/visual input and sleep remains unclear. To address this knowledge gap, we have started an investigatory survey of sleep behaviour using classic mutant lines to manipulate phototransduction and downstream neural transmission. We observed consistent day sleep fragmentation in flies with mutations in multiple phototransduction components. We also found hyperpolarised Drosophila photoreceptor resulted in shorter day sleep. We found a severe reduction in locomotor speed in several visual mutants during normal waking time preventing assessment of their sleep-linked immobility. In summary, we provide a rigorous quantification of several phototransduction genes and reveal the key role of visual input to promote sleep.

Hepatocyte nuclear factor 4 alpha (HNF4A) is a master regulator of hepatic differentiation and metabolism. Here, we identify and characterize a truncating Q164X mutation that impairs HNF4A transcriptional activity in vitro and causes embryonic lethality when homozygous. Functional assays revealed that the Q164X protein retains nuclear localization but exhibits severely reduced DNA binding and transcriptional activation. CRISPR-generated Q164X mice showed no viable homozygotes, confirming the essential role of HNF4A in early embryogenesis. Unexpectedly, heterozygous Q164X mutants displayed reduced liver tumorigenesis following diethylnitrosamine and high-fat diet treatment, despite downregulation of HNF4A target genes such as ApoB and Hnf1a . These results suggest that partial HNF4A deficiency may trigger compensatory metabolic networks that protect against carcinogenic stress. Collectively, our study establishes Q164X as a loss-of-function HNF4A mutation with paradoxical tumor-suppressive effects in vivo.

ABSTRACT RNAs move through the extracellular space to transmit information between cells, including mammalian neurons, yet how specific RNAs are channeled into these extracellular routes is unknown. Using genome-wide CRISPR screening, proteomics, and high-sensitivity transcriptomics in a neuronal cell line, we identify domesticated retroviral proteins and RNA- modifying enzymes that regulate RNA loading into and transportation via extracellular vesicles. We show that the pseudouridine synthase PUS1 is a key determinant of RNA trafficking, and that its catalytic product in RNA, pseudouridine, is both necessary and sufficient for extracellular RNA export. We further show that myosin light chain 6 (MYL6) is a pseudouridine-binding protein required for secretion of synthetic and endogenous RNAs. These findings reveal a biochemical code linking chemical RNA modification to extracellular transport, and establish a framework to study the function of extracellular RNAs in the nervous system and beyond.

Bacteria are frequently attacked by viruses, known as phages, and rely on diverse defence systems like restriction endonucleases and CRISPR-Cas to survive. While phages can evade these defences by covalently modifying their DNA, these non-canonical nucleobases create a strong selective pressure for host proteins that can recognize and exploit them. Here, using a structure-guided discovery approach, we identify widespread families of DNA glycosylases that protect bacteria against phages that incorporate modified guanine bases into their DNA. Despite high sequence variation, these enzymes share a conserved glycosylase fold and occur across bacterial lineages. We also uncover a distinct glycosylase superfamily that defends against phages with thymidine modifications, showing that glycosylases have repeatedly evolved as antiviral defences. Together, these findings reveal DNA glycosylases as versatile effectors of bacterial immunity and underscore structure-guided discovery as a powerful strategy for uncovering hidden layers of antiviral defence.

Abstract Next-generation sequencing (NGS) studies have identified > 200 potential genetic drivers of chronic lymphocytic leukemia (CLL). Nevertheless, the prognostic and functional impact of numerous mutations remains elusive. Here, we assessed the clinical and biological implications of ZMYM3 mutations in CLL, a gene recurrently mutated in 2–4% of patients. NGS analyses of 487 CLL cases identified 32 ZMYM3 variants, of which 75% were loss-of-function. Notably, 70% of ZMYM3 -mutated patients harbored mutations in the NOTCH signaling pathway, predominantly affecting NOTCH1 (60%). Clinically, ZMYM3 variants were associated with a shorter time to first treatment in univariate and multivariate analyses (median: 35 vs 52 months; p = 0.010), and stratified the clinical outcome of early-stage cases (median: 48 vs 91 months; p = 0.016). Functionally, CRISPR/Cas9 models demonstrated that ZMYM3 mutations cooperate with NOTCH1 mutations to induce profound transcriptional dysregulation. Furthermore, RNA-sequencing of cellular models and CLL patient samples revealed that ZMYM3 mutations impact DNA damage response and histone acetylation, leading to reduced chromatin accessibility. Additionally, ZMYM3 mutations promoted apoptosis evasion through caspase downregulation, correlating this anti-apoptotic phenotype with higher sensitivity to BCL-XL inhibition in vitro and ex vivo . Overall, this work underscores the clinical relevance of ZMYM3 mutations and provides novel insights into their contribution to CLL pathophysiology.

Abstract Understanding the intricate dynamics between host immunity and gut microbiota is fundamental for developing precision immunotherapies. However, existing tools lack the capacity to manipulate microbial genomes in a targeted, adaptive, and interpretable way while capturing downstream systemic effects. This gap limits the clinical translation of host–microbiome research, particularly in inflammatory and autoimmune diseases. The study aims to design and validate a CRISPR–AI integrated framework for real-time modulation of host immune responses through microbial gene editing. By dynamically targeting microbial determinants of host cytokine networks, the study seeks to optimize immunomodulatory outcomes in a programmable and biologically coherent manner. A hybrid methodological pipeline was implemented, combining CRISPR-Cas-based genome editing across 87 microbial strains with AI-driven modeling of host immune responses in 240 gastrointestinal tissue samples. Techniques included PLS-DA classification, Bayesian DAG-based causal inference, multivariate ANOVA, and dynamic feedback from cytokine expression. Gene loci MCR-21 and GNT-4B were targeted for immune optimization. A novel metric, the Biological Signal Integrity Score (BSIS), was introduced to quantify post-editing immunological coherence. IL-6 concentrations decreased by 52.4%, TNF-αby 46.1%, and IL-1β by 41.3% (all p < 0.001) following CRISPR editing of key microbial genes. Beneficial taxa such as Faecalibacterium prausnitzii increased by 2.7-fold, while harmful species like Enterococcus faecalis declined 3.1-fold. The model achieved 94.3% classification accuracy (PLS-DA) and 0.962 ROC-AUC for phenotype prediction. Causal inference identified 11 high-confidence edges (score > 0.85) linking microbial edits to cytokine cascades. BSIS reached 0.783, indicating high signal integrity post-editing. The article establishes a powerful cyber-biological framework to engineer host immune modulation by editing microbial genomes in response to real-time physiological feedback. The integration of CRISPR targeting, immune profiling, and AI-based optimization paves the way for next-generation precision therapeutics that are both adaptive and biologically grounded. The system enables recursive refinement, making it applicable to complex inflammatory conditions and personalized microbiome-based interventions.

Abstract Metastatic breast cancer (MBC) is a life-threatening disease with limited therapeutic options. The immune suppressive tumor microenvironment (TME) limits the potency of the antitumor immune response and facilitates disease progression and metastasis. Our current study demonstrates that p38α is a druggable target in the TME that regulates the outcome of the immune-tumor interaction. The study revealed that systemic blockade of p38α reduces metastasis, and this anti-metastatic response is negated by depletion of CD8 + T cells. Single-cell transcriptomic analysis of the immune-TME showed that pharmacological p38 inhibition (p38i) or tumor-specific inactivation of p38α by CRISPR/Cas9 (p38KO) resulted in a less exhausted and more activated CD8 + T cell phenotype. Immunophenotyping analyses demonstrated that p38 blockade reduced the expression of multiple inhibitory receptors on CD8 + T cells (i.e., PD-1, LAG-3, CTLA-4), indicating a reversal of immune exhaustion and enhanced immune activation systemically and in the TME. In contrast, p38 blockade did not exhibit inhibitory effects on T cells in proliferation assays in vitro and did not affect the proportion of regulatory T cells in vivo . The major negative impact of p38 blockade in vivo was on the myeloid populations, such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). Further, tumor p38α activity was required for the expression of cytokines/chemokines and tumor-derived exosomes with high chemotactic capacity for myeloid cells. Altogether, this study highlights a previously unrecognized the p38α-driven pathway that promotes an immune suppressive TME and metastasis, and that therapeutic blockade of p38α has important implications for improving antitumor immunity and patient outcomes.

Abstract Pea (Pisum sativum) has emerged as a major protein source for meat substitutes due to its high nutritional value, low production costs, and short life cycle. The generation of elite pea cultivars can be achieved via genetic engineering and CRISPR-based gene editing. However, this approach has lagged due to the low efficiency, lack of reproducibility, and cultivar dependency of the reported pea transformation protocols. Due to the challenges in the genetic engineering of pea, we employed a transient expression approach to identify optimal conditions for gene expression with the expectation that these conditions would enhance the efficiency of stable transformation. The highest transient expression was achieved when the Agrobacterium suspension was used at 1.6 optical density, combined with a co-cultivation time of one hour. With the optimized conditions and a staggered antibiotic selection protocol, genetic perturbations, including ectopic and antisense expression and CRISPR/Cas9 editing of the flavanone 3-hydroxylase (F3H) gene, were performed in a purple-seeded pea line. We report an efficient, stable transformation protocol for pea with a mean efficiency of 2.9%. Greenhouse-adapted seed-bearing transgenic plants were obtained in eight months. The T2 transgenic lines were verified using PACE-PCR and RT-qPCR analysis, which confirmed the transgenic status of the plant and altered expression of the F3H gene demonstrating successful genetic engineering in Pisum sativum .

Abstract Despite revolutionizing fungal genetic engineering, conventional CRISPR/Cas9-mediated knockouts rely on DNA double-strand breaks (DSBs), which can cause unwanted insertions and deletions, chromosomal abnormalities, and cytotoxicity. Base editors such as adenine base editors (ABEs), which convert A•T to G•C, and cytosine base editors (CBEs), which convert C•G to T•A, offer a safer alternative by enabling precise single-nucleotide changes without introducing DSBs. To overcome the limitations of traditional genome editing in filamentous fungi, we developed efficient base-editing systems in Aspergillus niger . For the first time, we constructed an ABE in A. niger , achieving up to 80% editing efficiency and inducing precise A-to-G mutations at conserved intron sites that disrupted gene function through mRNA mis-splicing. We also developed a highly efficient CBE system, capable of introducing premature stop codons with 50–100% efficiency. Furthermore, we established gene disruption approaches by targeting start codons: ABE-mediated A-to-G conversions (ATG-to-ACG and ATG-to-GTG) and CBE-mediated C-to-T conversion (ATG-to-ATA). To broaden the editing scope, we implemented a Cas9-NG variant recognizing a relaxed PAM sequence requiring only a single guanine (G), enabling editing at start codons and splice sites. Additionally, our base-editing systems enable multiplex gRNA delivery and marker-free editing of multiple genes. Together these improvements increase the number of genes targetable for disruption by base-editing in A. niger by 26.3% and enable near-complete coverage of 96% of the coding genes. Overall, this work demonstrates the potential of ABE and CBE systems as versatile, efficient, and safer alternatives to DSBs-based gene disruption in filamentous fungi.

Synaptic Ras GTPase-activating protein (SynGAP) regulates synaptic strength and neuronal signaling, with essential roles in cortical development and synaptic plasticity. Heterozygous loss-of-function variants in SYNGAP1 cause SYNGAP1 -related intellectual disability (SRID), a severe neurodevelopmental disorder characterized by epilepsy, developmental delay, and autism. SYNGAP1 mutations often result in haploinsufficiency, providing a strong rationale for gene-targeted therapies. However, no treatment currently addresses the underlying genetic cause of SRID. Here, we developed a CRISPR-mediated transcriptional activation (CRISPRa) approach to upregulate the functional Syngap1 allele in a SRID mouse model. CRISPRa activated Syngap1 , normalized SynGAP protein expression and downstream signaling, and rescued working memory deficits. We validated the translational potential of this strategy in human induced pluripotent stem cell (hiPSC)-derived excitatory cortical neurons. CRISPRa rescued SYNGAP1 in two distinct loss-of-function variant lines. Together, these findings demonstrate the feasibility of mutation-independent transcriptional activation as a therapeutic approach for SRID and its broader applicability to haploinsufficiency disorders.

ABSTRACT Precise regulation of Cas9 activity is essential to minimize off-target effects, mosaicism, chromosomal alterations, immunogenicity, and genotoxicity in genome editing. Although type II anti-CRISPR proteins (Acrs) can inhibit and regulate Cas9, their size and anionic charge generally prevent them from crossing the cell membrane. Existing Acr delivery methods employing vectors or electroporation are either slow and persistent or require external equipment, limiting their therapeutic utility. To address these challenges, we developed a cell-permeable Acr (6×NLS-Acr), which uses nuclear localization signals (NLSs) to cross the cell membrane. We conjugated 6×NLS-Acr to a fluorescent dye to elucidate its cellular entry mechanism and directly visualized its binding to a fluorescent Cas9·gRNA complex to study its inhibitory mechanism. 6×NLS-Acr (IC 50 = 0.47 µM) directly transduces human cells, including immortalized cell lines, embryonic stem cells, and 3D cell cultures, within 5 min, inhibiting up to 99% of Cas9 activity and increasing genome-editing specificity by nearly 100%. We further compared 6×NLS-Acr with our anthrax-derived Acr delivery platform. Our results demonstrate that 6×NLS-Acr is the most efficacious cell-permeable CRISPR-Cas inhibitor, significantly enhancing the precision and therapeutic potential of CRISPR-based genome editing.

Summary The stereoisomers of lactate, L- and D- are not only metabolic substrates but also signalling molecules, capable of activating and signalling through its G protein-coupled receptor, Hydroxycarboxylic acid receptor 1 (HCAR1). These stereoisomers are both produced by the gut microbiota at millimolar concentrations creating a physiological environment for lactate-sensing unique to the gut yet, poorly understood. Here we identify a role for D-/L-lactate on intestinal barrier function. A human colonic epithelial cell model, Caco2, activated Gαi signalling in response to both L- and D-lactate, although L-lactate exhibited a more potent and rapid Gi signal profile. When differentiated, apically but not basally treated D-/L-lactate enhanced tight junctions and reduced cell permeability, consistent with the apical localization of HCAR1. This improved barrier function occurred in a Gαi-dependent manner. In addition, apical lactate rescued the reduced intestinal barrier function induced by lipopolysaccharides. This work highlights the potential for D-/L-lactate supplementation in improving gut health.

Background: Cognitive, emotional, and social impairments are pervasive across neuropsychiatric conditions, where alterations in the tryptophan (Trp)–kynurenine pathway and its product kynurenic acid (KYNA) from kynurenine aminotransferases (KATs) have been linked to Alzheimer disease, Parkinson disease, depression, and post-traumatic stress disorder. In novel CRISPR/Cas9-engineered KAT II knockout (aadat-/- aka. kat2-/-) mice, we observed despair-linked depression-like behavior with peripheral excitotoxicity and oxidative stress. KAT II’s role and its crosstalk with serotonin, indole-pyruvate, and tyrosine (Tyr)–dopamine remain unclear. It is unknown whether deficits extend to cognitive, emotional, motor, and social domains or whether brain tissues mirror peripheral stress. Objectives: Delineate domain-wide behaviors, brain oxidative/excitotoxic profiles, and pathway interactions attributable to KAT II. Results: Behavior was unchanged across strains. kat2-/- deletion remodeled Trp metabolic pathways: 3-hydroxykynurenine increased, xanthurenic acid decreased, KYNA fell in cortex and hippocampus but rose in striatum, quinaldic acid decreased in cerebellum and brainstem. These region specific changes indicate metabolic stress across the brain and align with higher oxidative load and signs of excitotoxic pressure. Conclusion: Here we show KAT II deletion reshapes regional Trp metabolism and amplifies oxidative and excitotoxic imbalance. Although domain-wide behavioral measures, spanning cognition, sociability, and motor coordination, remained largely unchanged, these neurochemical alterations signify a latent emotional bias rather than overt depressive-like behavior. This work therefore refines prior findings by delineating KAT II–linked biochemical vulnerability as a potential substrate for stress-reactive affective dysregulation.

Nanobodies (single-domain antibodies, VHHs) have emerged as versatile tools for evaluating and treating Alzheimer’s disease (AD). They offer unique advantages over traditional antibodies and small molecules, including small size, stability, and specificity. In AD, nanobodies were used to neutralize toxic amyloid-β oligomers, inhibit tau generation and aggregation, and modulate neuroinflammation, thereby demonstrating significant therapeutic potential. The delivery of nanobodies requires advanced strategies, including intranasal and intrathecal routes, receptor-mediated transport, plasma protein binding with albumin, and focused ultrasound to facilitate brain penetration. Additionally, to improve nanobody delivery precision, half-life, and efficacy, strategies such as integrating nanobodies with nanoparticles, dendrimers, liposomes, and viral vectors are being employed. In fact, nanobodies are applied beyond monotherapy across multiple technological platforms to optimize brain delivery and target multiple targets. Nanobodies have been used on bispecific and trispecific antibody platforms, as well as in CRISPR/Cas9 editing and AI-driven technologies, to expand their applications. Recently, preclinical evidence has been mounting on the efficacy of nanobodies in clearing Aβ and tau, preserving synapses, and normalizing biomarkers. Notably, clinical trials of bispecific antibodies, including trontinemab, are signaling translational progress and regulatory approvals, and further support would validate this class of therapeutic molecules. This review critically delineates the current molecular mechanisms, emerging strategies, and delivery platforms, and emphasizes the potential of nanobodies as promising therapeutic and diagnostic molecules in AD therapeutics.

Clubroot disease, caused by Plasmodiophora brassicae, is a major global threat, causing severe yield losses of up to 100% in heavily infested fields. Interspecific hybridization is essential for the transfer of clubroot resistance genes among the Brassica species. This review aimed to describe the sources of clubroot resistance, categorize their types in Brassica crops, and identify the most effective techniques and underutilized sources for both intergeneric and interspecific hybridization. A systematic literature review served as the foundation for expert analysis, encompassing a comprehensive list of known sources of resistance and a detailed description of their characteristics, including monogenic, polygenic, dominant, and recessive traits. In addition, this review specifies techniques suitable for gene transfer, such as markers, embryo rescue, somatic hybridization, and CRISPR/Cas. Based on literature, underutilized directions for genetic crosses have been proposed. These conclusions suggest that combining biotechnological methods, including markers, CRISPR/Cas, and embryo rescue, with intergeneric crosses offers the potential to transfer resistance genes from previously untapped sources.

Loss of the prolyl endopeptidase-like (PREPL) protein causes congenital myasthenic syndrome-22 (CMS22), a rare neuromuscular and metabolic disorder. PREPL belongs to the serine hydrolase superfamily, but its physiological substrates remain unknown. Based on the predicted lipid binding pocket in its crystal structure and its in vitro esterase activity, we hypothesized that PREPL might act as a lipase in vivo and directly regulate lipid metabolism. To test this, we performed unbiased lipidomics in Prepl knockout (KO) mouse brains and CRISPR-Cas9-generated KO cell lines. Across tissue and cell types, global phospholipid composition was largely unchanged, with only modest, non-significant increases in lysophospholipids, arguing against a direct role of PREPL in (lyso)phospholipid turnover. In contrast, PREPL KO HEK293T cells exhibited a significant accumulation of triacylglycerols (TAGs) and an increased number of lipid droplets, indicating a selective shift toward lipid storage. Given the central role of peroxisomes in lipid metabolism, we assessed PREPL localization and examined peroxisome number, morphology, and levels of key peroxisomal proteins. PREPL did not localize to peroxisomes, and peroxisome number and proteins levels were largely unchanged. However, KO cells displayed elongated peroxisomes, a phenotype possibly linked to mitochondrial dysfunction. Indeed, previous studies have shown that PREPL localizes to mitochondria and is required for respiratory chain activity and oxidative phosphorylation. These mitochondrial defects are predicted to impair fatty acid β-oxidation and disrupt redox balance, thereby promoting TAG synthesis and lipid droplet biogenesis as adaptive responses. Overall, our findings indicate that PREPL does not act as a canonical lipase but indirectly alters lipid homeostasis through its critical role in mitochondrial function. Elevated TAG levels and altered peroxisome morphology likely represent secondary consequences of impaired mitochondrial fatty acid metabolism in PREPL-deficient cells. These results establish a mechanistic link between mitochondrial dysfunction and lipid remodeling in PREPL deficiency, providing novel insights into the metabolic pathology of CMS22.