SUMMARY While cortical organoids have been used to model different facets of neurodevelopmental conditions and human brain evolution, cerebellar organoids have not yet featured so prominently in the same context, despite increasing evidence of this brain regions importance for cognition and behavior. Here, we provide a longitudinal characterization of cerebellar organoids benchmarked against human fetal data and identify at very early stages of development a significant number of dynamically expressed genes relevant for neurodevelopmental conditions such as autism and attention deficit hyperactivity disorders. Then, we model an ASD mutation impacting CHD8, showing both granule cell and oligodendrocyte lineages prominently affected, resulting in altered network activity in more mature organoids. Lastly, using CRISPR/Cas9 editing, we also model an evolution-relevant mutation in a regulatory region of the CADPS2 gene. We investigate the effect of the derived allele exclusive to Homo sapiens, identifying a rerouting of the CADPS2-expression in rhombic lip cells, coupled with a different sensitivity to hypoxia which in turn lead to a differential timing of granule cell differentiation. HIGHLIGHTS Longitudinal characterization of cerebellar organoids uncovers disorder related genes especially at early stages of development Mutation in CHD8 alter rhombic lip and oligodendrocytes differentiation via WNT pathway Rerouting of CADPS2 expression, delaying differentiation and migration, in recent human evolution IN BRIEF Aprile and colleagues longitudinally profiled cerebellar organoids, benchmarking them against a fetal human atlas and identified a highly dynamic expression of genes related to cognitive and behavioral disorders especially at early stages of differentiation. Organoids were used to model the impact of a high-penetrance mutation associated with autism spectrum disorder and a high-frequency derived allele in Homo sapiens predicted to have played a role in recent brain evolution.

Although recent plant pan-genome studies have revealed extensive copy number variations, there are limited studies addressing the phenotypic consequences of these variations. Here, we manipulated the copy number of OsMADS18 in rice (Oryza sativa) through gene editing and evaluated the effects. OsMADS18, which encodes a MADS-box transcription factor known to regulate plant development, is tandemly duplicated in the elite rice cultivar ‘Hitomebore’ as a natural variant. We therefore edited the OsMADS18 copy number using a basic CRISPR/Cas9 system and established rice lines harboring one to three tandem copies of OsMADS18, as identified through high throughput qPCR screening. The presence of one to three OsMADS18 tandem copies was reflected in stepwise increases in transcript levels and concomitant trait values for leaf blade length and culm length. These results demonstrate that manipulating gene copy number can fine-tune important agronomic traits, providing a novel breeding strategy for crop improvement.

1-deoxy-sphingolipids (1-deoxySLs) are atypical sphingolipids synthesized by the serine palmitoyltransferase (SPT) when L-alanine is used instead of its canonical substrate L-serine. Increased 1-deoxySLs are associated with sensory neuropathies such as Hereditary Sensory and Autonomic Neuropathy type 1 (HSAN1) and diabetic polyneuropathy (DPN). Despite their known cellular, mitochondrial, and neurotoxic effects, the mechanisms underlying their toxicity remain poorly understood. Using a CRISPR interference (CRISPRi) screening approach, we identified CERS2, ELOVL1, ACACA, HSD17B12, and PTPLB as key mediators of 1-deoxySL-induced toxicity. All genes are integral to the biosynthesis of very long-chain (VLC) fatty acids and VLC-ceramides. We validated these findings through genetic knockdown experiments, cytotoxicity assays, and stable isotope-resolved lipidomics via LC-MS/MS. Pharmacological inhibition of ELOVL1 using a preclinical tested compound alleviated the cellular, mitochondrial, and neuronal toxicity induced by 1-deoxySLs. Supplementation experiments combining 1-deoxySLs with various VLC fatty acids revealed that 1-deoxyDHceramide conjugated to nervonic acid (m18:0/24:1) is the principal toxic specie. Further mechanistic studies showed that m18:0/24:1 induces apoptosis through the mitochondrial permeability transition pore (mPTP) formation. Inhibition of BAX or blocking mPTP formation with cyclosporin A effectively prevented toxicity. In conclusion, our findings demonstrate that 1-deoxyDHCeramides conjugated to nervonic acid are the primary mediators of 1-deoxySL toxicity, acting through mitochondrial dysfunction and BAX-dependent apoptosis.

Resistance to targeted therapy in BRAF-mutant melanomas can arise from drug-tolerant persister cells, which can non-genetically escape drug-induced quiescence to resume proliferation. To investigate how melanoma cells escape drug to re-enter the cell cycle within 3-4 days of BRAF and MEK inhibition, we computationally reconstructed single-cell lineages from time-lapse imaging data, linking key signaling pathways to cell-cycle fate outcomes. We found that ERK reactivation, while necessary, is insufficient for cell-cycle re-entry under MAPK inhibition. Instead, mTORC1 emerged as an additional critical mediator, enhancing translation and cell growth in cells destined for drug escape. We further found that ERK and mTORC1 signaling converge to produce Cyclin D1 protein levels, a key bottleneck for cell-cycle re-entry. Using CRISPR to tag endogenous Cyclin D1, we found that future escapees markedly upregulate Cyclin D1 at least 15 hours prior to drug escape, compared to non-escapees. Importantly, this early upregulation enables accurate prediction of future escapees from non-escapees, underscoring how differences in Cyclin D1 accumulation precede and govern the timing and likelihood of cell-cycle re-entry in persister cells. Our findings suggest that variability in ERK and mTORC1 activity underlies the heterogeneous Cyclin D1 levels observed, influencing the proliferative potential of persister cells and ultimately shaping the diverse cell-cycle behaviors observed under drug treatment. Cyclin D1 protein therefore emerges as both a critical biomarker and a therapeutic target for preventing cell-cycle re-entry in BRAF/MEK-treated melanoma. One Sentence Summary Cyclin D1 accumulation, driven by the integration of heterogeneous MAPK and mTORC1 signaling, is a critical bottleneck for cell-cycle re-entry in drug-treated melanoma cells.

ABSTRACT Large-cohort genome-wide association studies (GWAS) for alcohol use disorder (AUD) and AUD-related phenotypes have identified more than one hundred genetic loci. Functional study of those GWAS-identified loci might represent an important step toward understanding AUD pathophysiology. We found that genetic loci which are splicing quantitative trait loci (sQTLs) for the fibronectin III domain containing 4 ( FNDC4 ) gene in the brain were identified by GWAS for both AUD drug treatment outcomes and AUD risk. However, FNDC4 function in the brain and how it might contribute to AUD pathophysiology remain unknown. In the present study, we characterized GWAS locus-associated FNDC4 splice isoforms, studies which suggested that FNDC4 alternative splicing results in loss-of-function for FNDC4. We also investigated FNDC4 function using CRISPR/cas9 gene editing, and the creation of human induced pluripotent stem cell (iPSC)-derived neural organoids joined with single-nucleus RNA sequencing. We observed that knock-out (KO) of FNDC4 resulted in a striking shift in the relative proportions of glutamatergic and GABAergic neurons in iPSC-derived neural organoids, suggesting a possible important role for FNDC4 in neurogenesis. We also explored potential mechanism(s) of FNDC4 -dependent neurogenesis with results that suggested a role for FNDC4 in mediating neural cell-cell interaction. In summary, this series of experiments indicates that FNDC4 plays a role in regulating cerebral cortical neurogenesis in the brain. This regulation may contribute to the response to AUD pharmacotherapy as well as the effects of alcohol on the brain.

The human cerebellum is a specialized brain region that is involved in various neurological and psychiatric diseases but has been challenging to study in vitro due its complex neurodevelopment and cellular diversity. Despite the progress in generating neural tissues from human induced pluripotent stem cells (iPSCs), an organoid model that recapitulates the key features of cerebellar development has not been widely established. Here, we report the generation of a 60-day method for human cerebellar organoids (hCBOs) that is characterized by induction of rhombomere 1 (R1) cellular identity followed by derivation of neuronal and glial cell types of the cerebellum. In contrast to forebrain organoids with multiple neural rosettes and inside-out neuronal migration, hCBOs develop a SOX2+ cerebellar plate on the outermost surface of organoids with outside-in neuronal migration, which is a characteristic hallmark of cerebellar histogenesis. These hCBOs produced various other cell types including granule neurons, Purkinje cells, Golgi neurons, and deep cerebellar nuclei. By using a glial induction strategy, we generate Bergmann glial cells (BGCs) within the hCBOs that not only serve as scaffolds for granule cells migration but also enhance electrophysiological response of the hCBOs. Furthermore, by generating hCBOs from patients with Friedreich’s ataxia (FRDA), we revealed abnormal disease-specific phenotypes that could be reversed by histone deacetylase (HDAC) inhibitors and gene editing by CRISPR-Cas9. Taken together, our advanced hCBO model provides new opportunities to investigate the molecular and cellular mechanisms of cerebellar ontogenesis and utilize patient-derived iPSCs for translational research.

The chromosomal passenger complex (CPC; Borealin-Survivin-INCENP-Aurora B kinase) ensures accurate chromosome segregation by orchestrating sister chromatid cohesion, error-correction of kinetochore-microtubule attachments and spindle assembly checkpoint. Correct spatiotemporal regulation of CPC localization is critical for its function. Phosphorylations of Histone H3 Thr3 and Histone H2A Thr120 and modification-independent nucleosome interactions involving Survivin and Borealin contribute to CPC centromere enrichment. However, mechanistic basis for how various nucleosome binding elements collectively contribute to CPC centromere enrichment and whether CPC has any non-catalytic role at centromere remain open questions. Combining a high-resolution cryoEM structure of CPC-bound H3Thr3ph nucleosome with atomic force microscopy and biochemical and cellular assays, we demonstrate that CPC employs multipartite interactions involving both static and dynamic interactions, which facilitate its engagement at nucleosome acidic patch and DNA entry-exit site. Perturbing the CPC-nucleosome interaction compromises protection against MNase digestion in vitro, as well as the dynamic centromere association of CPC and centromeric chromatin stability in cells. Our work provides a mechanistic basis for the previously unexplained non-catalytic role of CPC in maintaining centromeric chromatin critical for kinetochore function.

Abstract Influenza virus infections can cause severe complications such as Acute Necrotizing Encephalopathy (ANE), which is characterised by rapid onset pathological inflammation following febrile infection. Heterozygous dominant mutations in the nucleoporin RANBP2/Nup358 predispose to influenza-triggered ANE1. The aim of our study was to determine whether RANBP2 plays a role in IAV-triggered inflammatory responses. We found that the depletion of RANBP2 in a human airway epithelial cell line increased IAV genomic replication by favouring the import of the viral polymerase subunits, PB1, PB2 and PA, and promoted an abnormal accumulation of some viral segments in the cytoplasm. In human primary macrophages, this corroborated with an enhanced production of the pro-inflammatory chemokines CXCL8, CXCL10, CCL2, CCL3 and CCL4. Then, using CRISPR-Cas9 knock-in for the ANE1 disease variant RANBP2-T585M, we demonstrated that the point mutation is sufficient to drive CXCL10 expression following activation downstream of RIG-I and leads to a redistribution of RANBP2 away from the nuclear pore. Together, our results reveal that RANBP2 regulates influenza RNA replication and nuclear export, triggering hyper-inflammation, offering insight into ANE pathogenesis.

Oculopharyngodistal myopathy (OPDM) is caused by CGG triplet repeat expansions in six genes. To explore the genetics and epigenetics of OPDM, we conducted CRISPR/Cas9-targeted resequencing of repeat regions in 89 patients. Repeat regions essentially comprised pure CGG expansions, but exhibited size variability, even within patients. Expanded LRP12 and GIPC1 alleles showed distinct single nucleotide variant patterns, suggesting founder haplotypes. LRP12 -expanded reads lacked flanking sequences present in non-expanded reads, whereas GIPC1 expanded repeats contained specific nucleotide patterns in their 5’-regions. Structural variations were identified in some patients. A significant inverse correlation was observed between repeat length and age at onset in patients with GIPC1 or NOTCH2NLC expansions, while this was disturbed by higher methylation of expanded regions in patients with LRP12 expansions, leading to delayed onset. These findings reveal a complex interplay among repeat size, sequence context, and epigenetic state in OPDM pathogenesis, advancing knowledge and providing opportunities for therapeutic intervention.

Resistance development is an inevitable failure mode of many drugs, pointing to the need to develop agents with orthogonal resistance mechanisms. Induced-proximity modalities, an emergent class of therapeutics, operate by forming a ternary complex with the protein-of-interest (POI) and effectors, unlike classical inhibitors that form binary complexes with the POI. Using KRAS as a model system, we employed base editor tiling mutagenesis screening to show that induced-proximity inhibitors exhibit orthogonal resistance mechanisms to classical inhibitors despite overlapping binding sites, offering an opportunity to circumvent resistance mechanisms of classical inhibitors. These findings highlight the use of base editor mutagenesis screens to prioritize inhibitors with orthogonal resistance mechanisms and the potential of induced-proximity inhibitors to overcome the drug resistance of classical inhibitors.

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.

Fever is a hallmark of malaria. Several studies have linked febrile temperatures to reduced parasite viability, but also to increased cytoadhesion, a key driver of pathology. However, different mechanisms have been proposed to cause changes in cytoadhesion and parasite sensitivity to heat. Here, we demonstrate that exposure of Plasmodium falciparum -infected red blood cells (iRBCs) to physiologically relevant febrile heat stress (39 °C), derived from patient data, enhances cytoadhesion through increased trafficking of the major virulence factor PfEMP1 to the iRBC surface. This phenomenon is not limited to PfEMP1 and common laboratory strains, as it extends to the surface nutrient channel PSAC in four clinical isolates of diverse geographic origin. The increased surface protein display occurs without changes in overall protein expression or parasite developmental progression. Using phosphoproteomics and proximity labelling, we find that elevated temperature also increases trafficking and phosphorylation of exported proteins into the RBC. Enhanced export is likely reliant on the presence of a transmembrane domain as shown by NanoLuc reporter assays. Collectively, our results indicate that febrile temperatures commonly experienced during infection can accelerate protein export, likely at the parasitophorous vacuole. This enhanced export following heat stress is relevant because increased cytoadhesion could influence disease severity through earlier iRBC sequestration and elevated bound parasite mass.

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.