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.

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.

Obligate intracellular bacterial pathogens cannot grow extracellularly, a trait that renders them highly understudied, but also endows them with unexpected host dependencies. Here, using the emerging obligate intracellular tickborne pathogen Rickettsia parkeri, we conducted a genome-scale CRISPR/Cas12a knockout infection screen to identify human cell determinants of rickettsial intracellular fitness. We discovered that the host peptidylprolyl isomerase cyclophilin A (PPIA/CypA), was essential for the formation of the R. parkeri actin tails that enable pathogen motility. PPIA localized to actin-associated bacterial poles and directly interacted with Sca2, the R. parkeri surface-exposed autotransporter chiefly responsible for actin tail nucleation. Sca2 bound PPIA through a domain implicated in surface translocation, and Sca2 failed to reach the R. parkeri surface in PPIA-deficient host cells. We propose that host PPIA enables Sca2 surface exposure during R. parkeri infection through a direct interkingdom protein maturation event, which represents an unexplored axis of the intracellular host-bacterial interface.

Abstract Memory engrams are formed by activity-dependent recruitment of distinct subsets of excitatory principal neurons (or neuronal ensembles) whereas inhibitory neurons pivot memory lability and stability. However, the molecular logic for memory engrams to preferentially recruit specific type of interneurons over others remains enigmatic. Using activity-dependent single-cell transcriptomic profiling in mice with training of cued fear memory and extinction, we discovered that neuropeptide Y (NPY)-expressing (NPY+) GABAergic interneurons in the ventral hippocampal CA1 (vCA1) region exert fast GABAergic inhibition to facilitate the acquisition of memory, but bifurcate NPY-mediated slow peptidergic inhibition onto distinct sub-ensembles underlying the extinction of single memory trace. Genetically encoded calcium and NPY sensors revealed that both calcium dynamics of NPY+ neurons and their NPY release in vCA1 ramp up as extinction learning progresses while behavioral state switches from “fear-on” to “fear-off”. Bidirectional manipulations of NPY+ neurons or NPY itself demonstrated that NPY is both necessary and sufficient to control the rate and degree of memory extinction by acting on two physically non-overlapping sub-ensembles composed of NPY1R- and NPY2R-expressing neurons. CRISPR/Cas9-mediated knockout of NPY2R or NPY1R further unravels that NPY co-opts its actions on these two sub-ensembles to gate early fast and late slow stages of extinction. These findings exemplify the intricate spatiotemporal orchestration of slow peptidergic inhibitions from single subtype of GABAergic interneurons to fine-tune engram lability verse stability of memory.

Abstract Aspergillus fumigatus ( AF ) is the predominant pathogen implicated in invasive aspergillosis (IA) in humans; therefore, prompt and accurate detection is critical for the effective prevention and management of IA. This study developed a rapid detection system targeting the AF -specific anxC4 gene by integrating enzymatic recombinase amplification (ERA) with CRISPR/Cas12a. The reaction proceeds at a stable temperature of 37°C, with amplification and detection systems separately positioned in the tube lid and bottom, respectively, effectively minimizing aerosol contamination typically associated with product transfers. To enhance sensitivity, the One Pot method was optimized. Consequently, the fluorescence detection limit reached 1 fg/µL, and the sensitivity of the test strip reached 10 fg/µL, with no cross-reactivity observed against other fungi. Detection of AF was completed within 60 min, and results were visually displayed through fluorescence signals and nucleic acid test strips. Clinical practicality was further evaluated using aspergillosis samples, which demonstrated satisfactory performance. Pure culture results confirmed that out of 62 sputum samples, 32 were positive and 30 negative. Evaluation of 62 clinical samples using the One Pot ERA-CRISPR/Cas12a system demonstrated sensitivity and specificity rates of 93.75% and 93.33%, respectively, via fluorescence detection, and 90.63% sensitivity and 96.67% specificity using lateral flow strips.

G protein-coupled receptors (GPCRs), the largest family of transmembrane proteins, transduce extracellular stimuli into intracellular signaling cascades to orchestrate human physiology. The transport of newly synthesized receptors from the endoplasmic reticulum (ER) to the plasma membrane (PM) determines cellular responsiveness to incoming ligands, yet the molecular machinery governing GPCR export remains incompletely defined. Here, we combine a synchronized cargo-release assay with a genome-wide CRISPR/Cas9 screen to systematically map regulators of GPCR ER-to-PM transport. Focusing on the δ-opioid receptor (DOR), a prototypical class A GPCR, we identify CNIH1 as a dedicated export factor. In the absence of CNIH1, DOR is retained intracellularly with immature glycosylation, and drives reduced PM signaling. CNIH1 localizes to both ER exit sites and the Golgi, promoting the anterograde transport of a subset of class A GPCRs. Opioid receptors directly interact with CNIH1 and require its putative COPII-binding site for export. Distinct from other human cornichon homologs, CNIH1 defines a selective GPCR-sorting receptor that couples GPCR biosynthesis to signaling competence.

ABSTRACT One of the seminal discoveries from genetic studies of autism spectrum disorder and related neurodevelopmental disorders (NDD) has been that loss-of-function (LoF) mutations in many genes that impact chromatin and transcriptional regulation confer substantial liability to NDD. Haploinsufficiency of the epigenetic regulator POGZ represents one of the strongest such associations; however, little is known about the direct or indirect regulatory targets of POGZ , or the mechanisms by which loss of this chromatin modifier alters early neuronal development and synaptic functions. Here, we created an allelic series of CRISPR-engineered human induced pluripotent stem cell (hiPSC) clones harboring mono- and biallelic POGZ deletions. In hiPSC-derived neural stem cells (NSC) and Neurogenin 2-induced neurons (iN), POGZ LoF altered the expression of genes associated with critical cellular processes and neuronal functions, including synaptic and intracellular signaling and extracellular matrix organization. Our multiomics profiling also showed altered footprinting of critical transcription factors (e.g., activator protein 1 complexes) that were enriched at promoters of differentially expressed genes associated with synaptic function. To further interrogate the shared molecular changes in neuronal development associated with NDD and POGZ regulation, we compared our results to deletions of the transcription factor MEF2C and the sodium channel gene SCN2A that we generated in these same isogenic iN. These analyses revealed strong enrichment of extracellular matrix and intracellular signaling disruption associated with POGZ and MEF2C deletion, whereas POGZ and SCN2A haploinsufficiency exhibited shared transcriptional effects on gene modules enriched for NDD-associated genes with opposing regulatory effects. Notably, we also observed alterations to synaptic firing rate and neurite extension with biallelic deletions, but not heterozygous lines, suggesting subtle effects in neuronal development associated with haploinsufficiency. Overall, these shared molecular consequences suggest key points of convergence that connect epigenetic regulation to neuronal function in the etiology of neurodevelopmental pathologies.

Abstract: Circadian clocks regulate daily rhythms in all eukaryotes through ∼24-hour transcriptional-translational feedback loops driven by clock proteins. However, the molecular mechanisms that set the 24-hour period remain poorly defined. Here, using single-molecule imaging and nascent RNA sequencing, we uncover an unexpected RNA-based molecular timer: a single intron in the Drosophila timeless ( tim ) gene that regulates circadian period length by controlling mRNA localization. Strikingly, we find that ∼50% of tim mRNAs are localized to the nucleus due to inefficient post-transcriptional splicing of a single intron (which we named intron P ), in contrast to other core clock transcripts that localize predominantly to the cytoplasm. CRISPR-mediated removal of intron P abolishes nuclear retention of tim transcripts, leading to accelerated TIM protein accumulation and a shortened ∼22-hour period with reduced rhythmic robustness. Remarkably, insertion of intron P alone into heterologous reporters is sufficient to promote nuclear retention in both Drosophila and human cells, acting as a conserved checkpoint that withholds transcripts in the nucleus until splicing is complete. Finally, we identify three RNA-binding proteins, two repressors (Hrb27C and Squid) and one activator (Qkr58E-2, a Sam68 homolog), that modulate intron P splicing in a rheostat-like manner. Together, these findings establish tim intron P as the first intron-based molecular timer in circadian clocks and reveal splicing kinetics as a critical regulatory layer in temporal gene expression programs, with broad implications for other processes such as development and immunity.

Simian immunodeficiency viruses (SIVs) have crossed from apes to humans at least four times, but only one event gave rise to the AIDS pandemic. The host barriers that pandemic HIV-1 group M ( major ) strains overcame to spread efficiently in humans remain poorly understood. To identify such barriers, we performed CRISPR-Cas9 screens driven by the replication efficiency of SIVcpz, the chimpanzee precursor of HIV-1. Guide RNA libraries targeting more than 500 human genes encoding potential antiviral factors were inserted into the replication-competent SIVcpz MB897 molecular clone, which is phylogenetically closely related to HIV-1 group M strains. Propagation in Cas9-expressing human SupT1 T cells significantly enriched for sgRNAs targeting ADAR, AXIN1, CEACAM3, CD72, EHMT2, GRN, HMOX1, HMGA1, ICAM2, CD72, IFITM2, MEFV, PCED1B, SGOL2, SMARCA4, SUMO1 and TMEM173 . These hits only partially overlapped with those identified in analogous HIV-1–based screens, indicating virus-specific restriction profiles. Functional analyses confirmed that IFITM2 (interferon-induced transmembrane protein 2), PCED1B (PC-esterase domain–containing protein 1B), MEFV (Mediterranean fever protein, pyrin/TRIM20), and AXIN1 (Axis inhibition protein 1), restrict replication of SIVcpz but not of HIV-1 group M strains in primary human CD4⁺ T cells. These findings reveal previously unrecognized host factors that limit SIVcpz replication in human cells and highlight barriers that HIV-1 likely overcame during its adaptation for pandemic spread. One Sentence Summary CRISPR screens with replication-competent SIVcpz identify human antiviral factors limiting efficient viral replication after zoonotic transmission.

Abstract Over the past two decades, considerable progress has been made in cataloguing genes and active chromatin elements in humans. However, despite these efforts, less than a quarter of genes have been assigned a function in the context of human disease, which limits our ability to interpret clinical genome sequencing results. In the field of inherited retinal diseases, 30–40% of cases remain genetically undiagnosed. This may be partially due to our limited understanding of the function of most retina-expressed genes, which prevents the correct interpretation of their sequence variations. In the effort of elucidating retinal gene function, we aimed at developing a protocol for in-vivo CRISPR-based gene knock-out perturbation in the mouse retina. This methodology can be useful to study retinal biology in health and disease, to investigate the effects of ablation of novel uncharacterized genes, and to study possible genetic modifiers of retinal phenotypes.

The advent of CRISPR/Cas9 genome editing technology has revolutionized cancer research by enabling precise, efficient, and versatile manipulation of genetic sequences implicated in oncogenesis and tumor progression. This review highlights the pivotal role of CRISPR/Cas9 in unraveling cancer biology, developing innovative therapeutic strategies, and advancing personalized medicine. Conventional cancer treatments such as chemotherapy, radiotherapy, and surgery, while effective, suffer from significant limitations including non-specific toxicity and resistance, necessitating the exploration of novel targeted approaches. CRISPR/Cas9 offers unprecedented capabilities for targeted gene editing, including correction of oncogenic mutations, silencing of tumor-promoting genes, and restoration of tumor suppressor function. Additionally, it facilitates the generation of patient-specific tumor models such as organoids and xenografts that can guide therapeutic decision-making. Current preclinical studies and early-phase clinical trials demonstrate the feasibility and promise of CRISPR-based therapies, although challenges such as off-target effects, efficient delivery, and ethical considerations must be carefully addressed. Emerging technologies including base and prime editing, improved delivery vectors, and RNA-targeting Cas enzymes are expanding the CRISPR toolbox for cancer therapeutics. Furthermore, novel applications targeting the tumor microenvironment and microbiome are gaining traction. In summary, CRISPR/Cas9 represents a transformative platform driving the future of precision oncology, offering hope for more effective, tailored cancer treatments.

CRISPR-Cas9 sex ratio distortion (SRD) systems can suppress insect populations by biasing progeny toward males, but realizing such systems requires reliable Cas9 expression from insect Y chromosomes. Here, we tested whether the spermatocyte-specific β Tub85D promoter can drive functional Cas9 expression when inserted on the Drosophila melanogaster Y chromosome. Using CRISPR-mediated homology-directed repair, we generated a Y-linked β Tub85D -Cas9-T2A-eGFP construct and compared its activity with an autosomal counterpart. Whereas autosomal β Tub85D -Cas9 induced strong male-biased sex ratios when paired with an X-poisoning gRNA, the Y-linked construct failed to distort sex ratios and exhibited approximately 2,000-fold reduction in Cas9 transcript abundance. Nonetheless, weak but detectable GFP fluorescence and Cas9 transcripts confirmed partial Y-linked promoter activity. These findings provide the first direct experimental evidence of meiotic sex chromosome inactivation (MSCI) acting on the Drosophila Y chromosome, revealing that meiotic promoters can remain weakly active despite strong repression. This work defines transcriptional limits of the Drosophila Y chromosome and informs the design of next-generation Y-linked gene drives for sustainable insect control.

Hepatosplenic T-cell lymphoma (HSTCL) is a rare and aggressive neoplasm associated with poor responses to standard chemotherapy regimens and low survival rates. No targeted therapies are available for HSTCL, and preclinical models to test new treatment options have not been established. The JAK-STAT signaling cascade is a key dysregulated pathway in HSTCL, and STAT5B N642H is the most frequent somatic mutation in the disease. Here, we report on newly established clonal, murine γδ T-cell lymphoma cell lines initiated and driven by oncogenic STAT5B N642H , which recapitulate key immunophenotypic features, gene expression profiles and typically low cytolytic activity of patient-derived human HSTCL cells. CRISPR-Cas9 mediated knockout demonstrated growth dependence on STAT5B N642H . Murine C15 cells were allo-engrafted intravenously into both immunodeficient and immunocompetent mice to model an aggressive HSTCL-like disease at high penetrance, with recipient mice displaying hepatosplenomegaly and destructive γδ T cell organ infiltration, including bone marrow and blood involvement. We identified the potential of JAK inhibition as a targeted treatment strategy for HSTCL, and found the clinically approved JAK inhibitor upadacitinib to display selective anti-tumor efficacy against STAT5B -mutated HSTCL cell lines in vitro, in vivo , and in primary HSTCL patient samples. Overall, we describe the first robust STAT5B-driven preclinical model resembling features of HSTCL in an immune competent setting. This tool is expected to accelerate the study of HSTCL disease mechanisms and the testing of novel therapies. Our data further present the JAK inhibitor upadacitinib as a promising targeted treatment option for STAT5B -mutated HSTCL.