Summary Calcium, as a cellular second messenger, is essential for plant growth. A tip-focused Ca 2+ gradient in polarized cells is considered to drive cell expansion. The cell wall polysaccharide pectin is a major Ca 2+ binding structure and Ca 2+ homeostasis is influenced by the cell wall architecture. LRR-extensin (LRX) proteins are extracellular regulators of cell wall development that are anchored in the cell wall by their extensin domain. The extensin-less LRX1ΔE14 variant of the root hair-expressed LRX1 of Arabidopsis induces a dominant-negative effect resulting in aberrant root hairs. In an effort to identify the underlying mechanism of the root hair defect caused by LRX1ΔE14 , we isolated a su ppressor of dominant- ne gative effect mutant, sune42 . It codes for the CATION CALCIUM EXCHANGER 4 (CCX4) that localizes to the Golgi apparatus and was shown to have Ca 2+ transport activity. A detailed investigation of the Ca 2+ dynamics revealed that LRX1ΔE14 coincides with a defect in tip-focused cytoplasmic Ca 2+ oscillation, and this effect is alleviated by the sune42 mutation. Additionally, reducing Ca 2+ availability influences the LRX1ΔE14 -induced root hair defect. We conclude that sune42 suppresses the root hair defect in LRX1ΔE14 through modulating cytoplasmic Ca 2+ dynamics, pointing at the importance of the Golgi apparatus for cellular Ca 2+ homeostasis.

Telomeric sequences vary across the tree of life and intimately co-evolve with telomere-binding protein complexes. However, the molecular mechanisms allowing organisms to adapt to new telomeric sequences are difficult to gauge from extant species. Here, we reprogrammed multiple yeast lines to human-like telomeric repeats to unveil their molecular and fitness response to novel telomeres. Initially, the exchange of telomere sequences resulted in genome instability, proteome remodelling and severe fitness decline. However, adaptive evolution experiments selected for repeated mutations that drove adaptation to the humanized telomeres. These consisted of the recurrent amplification of the telomere-binding protein TBF1 , by complex aneuploidies, or in repeated mutations that attenuate the DNA damage response. Overall, our results outline a response that defines the adaptive molecular landscape to novel telomeric sequences. Teaser Yeast cells adapt to novel telomeric variation through amplification of telomere-binding proteins and inactivation of a telomeric DNA damage response

ABSTRACT The decision of naïve T cells to differentiate into a specific Th cell subset after antigen encounter is a critical pivot point of the immune response and, when out of balance, can lead to autoimmunity, allergy or immunodeficiency. Th subset differentiation is determined by the expression of transcription factors including IRF4. We here describe a point mutation (IRF4 L116R ) in the DNA-binding domain of IRF4 leading to a dysregulation of CD4-T cell subsets and their functions. This point mutation does not alter overall protein expression. In sharp contrast to IRF4-null mice, neither the CD4/CD8 ratio nor T cell activation and memory is altered in naïve mice carrying the point mutation. However, IRF4 L116R T cells are reduced in their capability to differentiate into Th1, Th17 and Treg cells in a dose-dependent manner, contrasting with the findings in IRF4 KO T cells. Particularly striking is the loss of Th1-differentiation in T cells from homozygous I rf4 L116R/L116R mice while T cells completely lacking IRF4 show no reduction in Th1 differentiation. Furthermore, despite maintained ability to generate Th17 cells, expression of the IRF4 L116R variant protects mice against disease development in the Th17 cell-dependent EAE mouse model of neuroinflammation. This contrasts with IRF4 knock-out mice, where expression of one wild-type allele of IRF4 is sufficient for full disease development. Together our results show that the L116R point mutation in IRF4 can selectively alter the differentiation and function of some CD4 T cell subsets and suggest that the L116R mutation is not a classical loss- or gain-of-function variant.

Anaplastic thyroid cancer (ATC) is the most aggressive form of thyroid cancer. Despite recent advances in treating BRAFV600E-driven ATC, therapy resistance remains a significant challenge, often resulting in disease progression and death. Leveraging a focused CRISPR/KO screen in parallel with a CRISPR/activation screen, both tailored on response to BRAFV600E inhibitor treatment, we identified TAZ (encoded by the WWTR1 gene) deficiency as synthetically lethal with BRAF inhibitor in ATC. TAZ is overexpressed in ATC compared to well-differentiated thyroid tumors. We demonstrate that TAZ-deficient ATC cells display heightened sensitivity to BRAF inhibitors both in vitro and in vivo . Using gene essentiality score across a large panel of cancer cell lines, we found that BRAFV600E-driven cancers are highly sensitive to TAZ loss, unlike their counterparts with wild-type BRAF and non-BRAFV600E. Mechanistically, we demonstrate that dabrafenib triggers the Unfolded Protein Response (UPR) under ER stress and suppresses protein synthesis. TAZ loss represses the UPR, reverses the inhibition of protein synthesis, and triggers increased cell death by ferroptosis in dabrafenib-treated ATC. Collectively, our findings unveil TAZ as a new target to overcome resistance to BRAF inhibitors in undifferentiated thyroid cancer.

Convergent evolution, the repeated evolution of similar phenotypes in response to the same selective pressures across multiple lineages, is widespread in nature. The extent to which the same genetic mechanisms contribute to convergent evolution could reveal whether the pathway towards these optimal endpoints is flexible or constrained to follow a particular route. Although mimicry of aposematic colour patterns is well known in Lepidoptera, our knowledge of the genetic basis of these convergent patterns is mostly restricted to a few closely-related species. Here we study the genetic basis of mimicry across seven species of Ithomiini and Heliconius butterflies and a day-flying Chetone moth, representing lineages that diverged between ∼1-120 Mya, each presenting similar colour pattern switches. In all the butterfly species, the genetic variants most strongly associated with convergent colour pattern switches are similarly located in non-coding regions near the genes ivory and optix . Colour pattern variation in the moth is associated with a ∼1 Mb inversion around ivory paralleling the supergene architecture of the co-mimic Heliconius numata . In contrast to previous studies in Heliconius , there is limited evidence of alleles shared by means of hybridization in convergence among closely-related ithomiine species. Repeated parallel evolution of regulatory switches via reuse of the same two genes suggests that convergent colour pattern evolution is highly constrained, even across large evolutionary timescales.

Abstract We performed a comprehensive meta-analysis of four major medical innovations (2015–2025) CRISPR gene editing, mRNA vaccines, AI diagnostics, and telemedicine focusing on clinical efficacy, health outcomes, and adoption. For CRISPR therapies in hemoglobinopathies, pooled data from six trials (115 patients) show robust clinical benefits: significant fetal hemoglobin induction, transfusion independence in β-thalassemia, and reduced sickle crises【1,2】. mRNA COVID-19 vaccines exhibited extremely high efficacy; pooled vaccine effectiveness was ~96% (95% CI 93–98%) after two doses【3】, far exceeding traditional platforms. AI diagnostic tools demonstrated moderate accuracy: a meta-analysis of 83 studies found mean diagnostic accuracy ~52% statistically comparable to physicians overall (no significant difference) but lower than expert clinicians【4】. Telemedicine interventions produced modest but positive effects: in a meta-review of 33 RCTs, telemedicine outcomes were at least as good as usual care (Cohen’s d≈0.21) across diverse conditions【5】. Notably, telehealth adoption surged during COVID-19 (e.g. a 683% increase in tele-visits at one center【6】). Comparative analysis indicates mRNA vaccines delivered the largest population-level impact (via prevention of disease), while CRISPR offers potentially curative benefits in niche populations. AI and telemedicine have improved diagnostic and care delivery processes with varying effect sizes. Our findings underscore each innovation’s significant but distinct contribution to global health.

Abstract Leishmania is a protozoan parasite causing leishmaniasis, a disease affecting millions globally. It is transmitted through bites from infected sand-flies, primarily of the Phlebotomus spp. or Lutzomyia spp. The parasite has two main life stages: the promastigote, found in the insect vector, and the amastigote, residing in the macrophages of the host. The mechanisms behind stage differentiation are not well understood. This study shows that redox metabolism modulations are crucial for the life cycle of Leishmania infantum, influencing stage transitions. Inhibiting redox metabolism caused significant morphological changes, from flagellated promastigotes to amastigote-like forms. These changes were evidenced by transcriptomic and metabolomic analyses. RNA sequencing indicated that redox inhibition affected several genes, including one for an iron transporter, LINF_310039600. Using CRISPR-Cas9, knockout mutants of this gene were created, revealing upregulation of amastin, a marker of the amastigote form, underscoring the role of redox metabolism in stage differentiation.

Abstract Pre-harvest sprouting (PHS), where seeds germinate on panicles before harvest under humid conditions, is a serious global issue in cereal crop production, including rice. OsERF94 was previously identified as a candidate gene associated with PHS through a genome-wide association study. In this study, we investigated the role of OsERF94 in PHS using CRISPR/Cas9 gene editing. The CRISPR/Cas9-mediated mutagenesis of OsERF94 induced frameshift mutations, resulting in a loss-of-function of OsERF94 in the 1-I-ET and 2-D-ET lines. The 1-I-ET and 2-D-ET lines exhibited significantly higher germination rates under PHS conditions compared to the wild type, indicating increased susceptibility to PHS. Whole-genome re-sequencing confirmed that few or no mutations could be detected at off-target candidate sites in both edited lines, ensuring the precision of the CRISPR/Cas9 gene editing. A transcriptome analysis revealed that OsERF94 modulates the expression of key GA biosynthetic and catabolic genes, including OsLOL1 , OsKO3 , OsGA3ox2 , and OsGA2ox5 , during both seed development and the early germination stages of PHS. The up-regulation of GA biosynthetic genes and the down-regulation of GA deactivation genes in both gene-edited lines likely led to elevated endogenous GA levels at 0 and 1 days after PHS, promoting germination under PHS conditions. These findings suggest that OsERF94 acts as a negative regulator of germination by modulating both GA biosynthesis and deactivation. Our findings contribute to expanding our knowledge of the molecular mechanisms of OsERF94 in PHS and highlight OsERF94 as a promising target for the genetic improvement of PHS resistance in rice-breeding programs.

Poison exons (PE) are highly conserved exon cassettes whose inclusion creates premature termination codons (PTCs) and triggers nonsense-mediated decay (NMD) of the mature transcript, therefore reducing protein expression. Despite their important role in post-transcriptional regulation, PEs remain poorly annotated due to the lack of systematic, transcriptome-wide approaches. In this study, we comprehensively investigated the function of PEs in the human brain and assessed the impact of pathogenic variants on their splicing. A comparative analysis of 957 eukaryotic transcriptomes revealed that humans exhibit the highest enrichment of NMD-targeted transcripts. To delineate the full landscape of PEs in the human transcriptome, we considered conserved (PhyloP score ≥ 20) alternatively spliced exons, less than 300 base pairs (bp) long, with the ability to introduce PTCs positioned more than 150 base pairs (bp) downstream of the transcription start site and outside the last two exons of the transcript. Our analysis identified 12,014 PEs in the human genome. For each PE, we calculated the percent-spliced-in (PSI) value across tissue types and developmental stages using GTEx and BrainSpan RNA-seq dataset, respectively. Overall, we identified 117 PEs uniquely found in the human brain, 1,214 PEs with brain-specific differential splicing compared to other tissues, and 1,610 PEs which splicing change during brain development. Integrating ClinVar and SpliceAI, we identified 1,877 annotated pathogenic variants predicted to affect the splicing of 891 PEs in the human brain. Notably, many of these variants were associated with neurodevelopmental and neurodegenerative disorders such as epilepsy, intellectual disability and frontotemporal dementia. We functionally validated the impact of selected variants using an optimized CRISPR prime-editing workflow in human cell lines. Our findings highlight PEs as pivotal regulators of gene expression in the human brain and establish a foundation for therapeutic strategies targeting PE mis-splicing in neurological diseases.

The two main cell types in the striatum, dopamine receptor 1 and adenosine receptor 2a spiny projection neurons (D1-SPNs and A2A-SPNs), have distinct roles in regulating motor- and reward-related behaviors. Cre-selective CRISPR-dCas9 systems allow for cell-type specific, epigenomic-based manipulation of gene expression with gene-specific single guide RNAs (sgRNAs) and have potential to elucidate molecular mechanisms underlying striatal subtype mediated behaviors. Conditional transgenic Rosa26:LSL-dCas9-p300 mice were recently generated to allow for robust expression of dCas9-p300 expression with Cre-driven cell-type specificity. This system utilizes p300, a histone acetyltransferase which regulates gene expression by unwinding chromatin and making that region of the genome more accessible for transcription. Rosa26-LSL-dCas9-p300 mice were paired with Drd1-Cre and Ador2a-Cre mice to generate Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 mouse lines and underwent behavioral phenotyping when sgRNAs were not present. Both Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 have cell-type specific expression of spCas9 mRNA. Baseline behavioral assessments revealed that, under a sgRNA absent nontargeted state, Drd1-Cre:dCas9-p300 mice display repetitive spinning behavior, hyperlocomotion and enhanced acquisition of reward learning in comparison to all genotypic littermates. In contrast, Ador2a-Cre:dCas9-p300 do not exhibit any changes in behavior in comparison to their littermates. Electrophysiological recordings of dorsal striatum D1-SPNs revealed that Drd1-Cre:dCas9-p300 mice have increased input resistance and increased spontaneous excitatory postsynaptic current amplitude, together suggesting greater excitatory drive of D1-SPNs. Overall, these data demonstrate the necessity to validate CRISPR-dCas9 lines for research investigations. Additionally, the Drd1-Cre:dCas9-p300 line has the potential to be used to study underlying mechanisms of stereotypy and reward-learning. Significance Statement Using CRISPR-based tools to identify cell-type specific epigenomic and transcriptional mechanisms in disease and behavior has high utility for the neuroscience field. Previous limitations related to implementation of CRISPR-editing systems in mice were thought to be overcome by the generation of transgenic mouse lines, including a novel Cre-dependent dCas9-p300 mouse line. Our data shows however that Drd1-Cre:dCas9-p300 mice, generated from breeding Drd1-Cre mice with the dCas9-p300 mice, have cellular and behavioral disruptions under a nontargeted sgRNA absent state. Overall, these data suggest caution in employing CRISPR-dCas9 systems, particularly transgenic mouse lines, for research investigations.

CRISPR-Cas genome editing toolkits have expanded the scope of genetic studies in various emerging model organisms. However, their applications are limited mainly to knockout experiments due to technical difficulties in establishing knock-in strains, which enables in vivo molecular tagging-based experiments. Here, we investigated knock-in strategies in the harlequin ladybug Harmonia axyridis , a model insect for evolutionary developmental biology, which shows more than 200 color pattern variations within a species. We tested several knock-in strategies using synthetic DNA templates. We found that ssDNA templates generated founder knock-in strains efficiently (2.5-11%), whereas the 5’ regions of ssDNA templates were frequently scraped when the insert length exceeded ∼40 bases. To overcome this limitation, we designed several 3’ extended DNA templates. Of those, fast-annealed 3’-extended double-stranded DNA templates showed extremely high founder generation efficiency (32-67%) and accuracy (67-100%). This strategy is also applicable to the two-spotted cricket Gryllus bimaculatus , demonstrating that the fast-annealed 3’-extended dsDNA template is a highly potent and versatile DNA template for generating knock-in strains in emerging model insects for developmental genetic studies. Summary statement Fast-annealed 3’-extended dsDNA templates facilitate efficient CRISPR-Cas9-mediated knock-in in emerging model insects.

ABSTRACT The human malaria parasite Plasmodium falciparum ( Pf ) expresses ten thrombospondin type 1 repeat (TSR) domain-bearing proteins at different stages throughout its life cycle. TSRs can be modified by two types of glycosylation: O-fucosylation at conserved serine (S) or threonine (T) residues and C-mannosylation at conserved tryptophan (W) residues. Pf TRAP, which is expressed in mosquito-stage sporozoites, has one TSR domain that is O-fucosylated at Thr 256 and C-mannosylated at Trp 250 . We employed site-directed mutagenesis by CRISPR/Cas9 gene editing to generate two Pf TRAP glyco-null mutant parasites, Pf TRAP_T256A and Pf TRAP_W250F. The fitness of these mutant parasites across the life cycle was compared to the parental NF54 wild type as well as a Pf TRAP knockout line. The glyco-null parasites exhibited major fitness defects comparable to knockout: sporozoites were unable to productively colonize the salivary glands and were highly impaired in gliding motility and the ability to invade cultured human hepatocytes. Protein analysis revealed significantly reduced Pf TRAP abundance in the glyco-null mutants despite normal transcript levels. These findings demonstrate that glycosylation of Pf TRAP’s TSR is critical for its proper expression and function, and underscores the importance of TSR glycosylation in the mosquito stage of the life cycle.

Enterococcus faecalis is a gram-positive bacterium and a common cause of hospital-associated infections. Three major CRISPR loci have been discovered in this species, namely CRISPR1-cas, CRISPR2 and CRISPR3-cas. We developed novel primers which target the CRISPR1-cas loci in E. faecalis and tested these primers on 26 E. faecalis isolates isolated from diverse settings from Segamat, Malaysia. Half of the isolates were found to carry the CRISPR1-cas9 locus, and the CRISPR1 array was successfully amplified in 12 out of 13 isolates that contained the cas9 gene. Characterisation of the CRISPR array shows that CRISPR1-cas shares similar array length and typical repeat sequences with CRISPR2 but differ significantly in terms of spacer identities and terminal repeat (TR) sequences. Most CRISPR spacers encode for chromosomal DNA sequences. Genotype characterisation based on ancestral spacer (AS) and TR sequences indicate that E. faecalis with the same CRISPR1-AS genotype do not always harbour same CRISPR2-AS genotypes, and vice versa. A combined CRISPR1-cas and CRISPR2 typing offers comparable discriminatory power to multilocus sequence typing (MLST), suggesting its potential to be used in short-term strain identification and epidemiological surveillance at a lower sequencing cost. Our study provides a genetic reference for future studies in the Southeast Asia region.

Synthetic biology applied to baculoviruses enables genome optimization through the targeted deletion of nonessential genes, enhancing recombinant protein expression. In this study, the CRISPR/Cas9 system with two tandem sgRNAs was used and validated as an efficient strategy to remove independently large genomic fragments from AcMNPV, all encoding proteins dispensable for budded virus production. The resulting mutant viruses were evaluated for their ability to express eGFP and HRPc in Sf9 cells and in Rachiplusia nu and Spodoptera frugiperda larvae. Deletions of the Ac15–Ac16 and Ac129–Ac131 regions led to significant increases in protein expression in infected cells and in larvae. In contrast, deletion of the Ac136–Ac138 region enhanced expression only in cultured cells but had a negative impact on larvae. Removal of the Ac148–Ac150 fragment caused a marked reduction in expression in both experimental systems. These findings confirm that although some genes are nonessential for systemic infection, their combined deletion can differentially affect recombinant protein expression depending on the host. This study validates not only an effective strategy for developing minimized baculovirus genomes but also the use of dual-guide CRISPR/Cas9 editing as a rapid and precise tool for genome engineering in baculovirus-based expression systems.

Adipose inflammation plays a key role in obesity-induced metabolic abnormalities. Epigenetic regulation, including DNA methylation, is a molecular link between environmental factors and complex diseases. Here we found that high fat diet (HFD) feeding induced a dynamic change of DNA methylome in mouse white adipose tissue (WAT) analyzed by reduced representative bisulfite sequencing. Interestingly, DNA methylation at the promoter of estrogen receptor α ( Esr1 ) was significantly increased by HFD, concomitant with a down-regulation of Esr1 expression. HFD feeding in mice increased the expression of DNA methyltransferase 1 ( Dnmt1 ) and Dnmt3a, and binding of DNMT1 and DNMT3a to Esr1 promoter in WAT. Mice with adipocyte-specific Dnmt1 deficiency displayed increased Esr1 expression, decreased adipose inflammation and improved insulin sensitivity upon HFD challenge; while mice with adipocyte-specific Dnmt3a deficiency showed a mild metabolic phenotype. Using a modified CRISPR/RNA-guided system to specifically target DNA methylation at the Esr1 promoter in WAT, we found that reducing DNA methylation at Esr1 promoter increased Esr1 expression, decreased adipose inflammation and improved insulin sensitivity in HFD-challenged mice. Our study demonstrates that DNA methylation at Esr1 promoter plays an important role in regulating adipose inflammation, which may contribute to obesity-induced insulin resistance.

ABSTRACT Phenotypic plasticity occurs when a genotype can produce more than one phenotype under different environmental conditions. Genetic accommodation allows plastic phenotypes to be tuned to new environments and eventually to even be lost via canalization/assimilation. The colourful and toxic Heliconiini butterflies have biochemical plasticity – they either sequester their cyanogenic glucosides (CG) from their larval hostplant or biosynthesize them when compounds for sequestration are not available. Here, we traced the evolution of CG biosynthesis in Heliconiini butterflies, a fundamental component of this biochemical plasticity. We first CRISPR-edited the CYP405s , which encode the first enzyme of this pathway in Heliconius erato, and confirmed that CYP405 -knockout caterpillars do not biosynthesize CGs. Then, we identified the CYP405 s and CYP332 s (the second gene in this pathway) in 63 species of the subfamily Heliconiinae, as well as in other lepidopterans. Most lepidopterans have a CYP332 , but CYP405 is mostly found in butterflies and has been duplicated in all Heliconius species. Both genes were independently co-opted into CG biosynthesis in the Heliconiinae butterflies and Zygaena moths, in which they were first functionally characterized. Finally, we performed ancestral reconstruction analyses of biochemical plasticity using data from over 700 Heliconiini butterflies. We demonstrated that although plasticity was ancestral in the whole tribe and allowed these butterflies to increase their hostplant range, it might have been lost in few specialized clades, such as the Sapho clade that just sequester CG from their hostplants. This clade has several CYP405 copies, but they lack structurally important P450 domains, which may explain why biosynthesized CGs are virtually lost in species of the Sapho clade. Our findings represent one of the few examples of plasticity-first evolution in which the genetic mechanisms associated with its accommodation/assimilation are known. This study also emphasizes the importance of co-option in the evolution of complex traits, such as toxicity.

Genome-wide association studies (GWAS) have identified numerous loci linked to late-onset Alzheimer’s disease (LOAD), but the pan-brain regional effects of these loci remain largely uncharacterized. To address this, we systematically analyzed all LOAD-associated regions reported by Bellenguez et al. using the FILER functional genomics catalog across 174 datasets, including enhancers, transcription factors, and quantitative trait loci. We identified 42 candidate causal variant-effector gene pairs and assessed their impact using enhancer-promoter interaction data, variant annotations, and brain cell-type-specific gene expression. Notably, the LOAD risk allele of rs74504435 at the SEC61G locus was computationally predicted to increase EGFR expression in LOAD related cell types: microglia, astrocytes, and neurons. Functional validation using promoter-focused Capture C, ATAC-seq, and CRISPR interference in the HMC3 human microglia cell line confirmed this regulatory relationship. Our findings reveal a microglial enhancer regulating EGFR in LOAD, suggesting EGFR inhibitors as a potential therapeutic avenue for the disease.

Retinoic acid (RA) is a transcriptional control agent that regulates several aspects of eye development including invagination of the optic vesicle to form the optic cup, although a target gene for this role has not been previously identified. As loss of RA synthesis in Rdh10 knockout embryos affects the expression levels of thousands of genes, a different approach is needed to identity genes that are directly regulated by RA. Here, we combined ChIP-seq for epigenetic marks with RNA-seq on eye tissue from wild-type embryos and Rdh10 -/-embryos that exhibit failure in optic cup formation. We identified a small number of genes with decreased expression when RA is absent that also have decreased presence of a nearby epigenetic gene activation mark (H3K27ac). One such gene was Alx1 that also has an RA response element (RARE) located near the RA-regulated H3K27ac mark, providing strong evidence that RA directly activates Alx1 . In situ hybridization studies showed that Rdh10 -/-embryos exhibit a large decrease in eye Alx1 expression. CRISPR/Cas9 knockout of Alx1 resulted in a defect in optic cup formation, thus demonstrating that RA directly activates Alx1 in order to stimulate this stage of eye development. Highlights The RA requirement for optic cup formation was examined using Rdh10 knockout embryos. Eye RNA-seq and ChIP-seq (H3K27ac) identified Alx1 as a potential RA target gene. Alx1 exhibits RA-regulated H3K27ac deposition near exon 1 associated with a nearby RARE. Alx1 knockout embryos display a misfolded optic cup with a ventral defect similar to Rdh10 KO.

Cerebral small vessel disease is a leading cause of cognitive decline and stroke in the elderly, with cerebral microbleeds (CMBs) as one of the key imaging biomarkers. Our understanding of its pathophysiology remains limited due to the lack of appropriate animal models. We report a novel mouse CMB model created by disrupting collagen IV, a core component of the vascular basement membrane (BM), specifically within brain microvessels. Targeted deletion of Col4a1 was achieved in adult mice using brain endothelial-specific AAV vectors with CRISPR/Cas9. MRI revealed numerous CMBs with distributions similar to those of human CMBs. CMB burden increased progressively over six months following Col4a1 deletion in a dose-dependent manner, accompanied by cognitive decline and motor incoordination. Histological examination revealed hemosiderin deposits corresponding to MRI-detected CMBs without evidence of macroscopic hemorrhage or white matter lesions, while ultrastructural analysis demonstrated significant BM thinning in Col4a1 -depleted microvessels. Analysis of human MRI and genomic data identified significant associations between CMB susceptibility and genetic variants in TIMP2 , an endogenous inhibitor of the matrix-degrading enzyme MMP2, underscoring the clinical relevance of our model. These findings establish a direct causal relationship between microvessel COL4A1 and CMB, suggesting that dysregulated collagen IV homeostasis in BM underlies CMB development.

SUMMARY Despite the availability of RAS inhibitors and the dependence of >90% of pancreatic ductal adenocarcinomas (PDAC) on oncogenic KRAS mutations, resistance to KRAS inhibition remains a serious obstacle. We show here that phosphoinositide 3-kinase (PI3K) plays a major role in this resistance through upstream activation of wild-type RAS signaling – beyond its known KRAS effector function. Combining proximity labeling, CRISPR screens, live-cell imaging, and functional assays we found that PI3K orchestrates phosphoinositide-mediated GAB1 recruitment to the plasma membrane, nucleating assembly of RAS signaling complexes that activate mitogen-activated protein kinase (MAPK) in an EGFR/SHP2/SOS1-dependent manner. We further demonstrate that inhibiting PI3K enhances sensitivity to mutant-specific KRAS inhibitors in PDAC cells, including cells with clinically identified PIK3CA mutations. Our findings refine RAS-PI3K signaling paradigms, reveal that PI3K-driven wild-type RAS activation drives resistance to KRAS inhibition, and illuminate new avenues for augmenting KRAS-targeted therapies in PDAC.

SUMMARY Energy expenditure (EE) is essential for metabolic homeostasis, yet its central regulation remains poorly understood. Here, we identify arcuate Kiss1 neurons as key regulators of EE in male mice. Ablation of these neurons induced obesity, while their chemogenetic activation increased brown adipose tissue (BAT) thermogenesis without affecting food intake. This action is mediated by glutamatergic projections from Kiss1 ARC neurons to CART/Lepr-expressing neurons in the dorsomedial hypothalamus, which activate the raphe pallidus-BAT pathway. CRISPR-mediated deletion of the vesicular glutamate transporter 2 ( Vglut2) from Kiss1 ARC neurons replicated the obesogenic effect. Furthermore, deletion of the melanocortin 4 receptor (MC4R) from Kiss1 neurons resulted in obesity, reduced energy expenditure and impaired thermogenesis. Optogenetic stimulation of pro-opiomelanocortin (POMC) fibers evoked inward currents in Kiss1 neurons, that were attenuated by MC4R antagonism. Our findings reveal a previously unrecognized neural circuit that mediates melanocortin action on energy expenditure, offering new insights into central mechanisms of metabolic control.

Abstract Active transport of chemical species across the cell membrane represents a critical biological and biotechnological function, allowing the cell to selectively import compounds of nutritional value whilst exporting potentially toxic compounds. Major facilitator superfamily (MFS) transporters represent a ubiquitous class able to uptake and export an array of different chemical species. When designing biosynthetic pathways within microbial hosts, for production or remediation, transport is often critical to the efficiency of the resulting engineered strain. However, transport is a commonly neglected node for characterisation and engineering given difficulties in producing, purifying and assaying membrane transport proteins outside of their native environment. Here, using syntenic analysis and genetically encoded biosensors a library of MFS transporters were screened for their ability to uptake the aromatic acids, protocatechuic acid and terephthalic acid. The structure activity relationships of the corresponding transporters, PcaK and TphK, were then assessed with library of aromatic acid effectors. Finally, the feasibility of protein engineering was assessed, by the creation of chimeric MFS transporters, revealing a degree of effector recognition plasticity and the modularity of core transmembrane domains. This study provides a library of validated MFS transporters and demonstrates the value of employing genetically encoded biosensors in the characterisation and engineering of this important transport function.

Huntington’s disease (HD) is a monogenic autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in the first exon of the HTT gene, yielding a gain-of-toxic-function mutant Huntingtin protein mHTT. CRISPR/Cas9 is a potentially powerful therapeutic tool for treating HD by eliminating mutant HTT (m HTT ) gene. We developed a specific SaCas9 guide RNA to target human m HTT , and a self-inactivating gene editing system that abolishes SaCas9 after a short transient expression for high gene editing efficiency and maximal safety to prevent off-target effects. Both conventional and the new self-inactivating gene editing systems achieved successful elimination of m HTT gene, 60-90% mHTT protein and 90% of mHTT aggregation in BAC226Q HD mouse brains, which resulted in significant long-term rescue of neural pathology, motor deficits, weight loss and shortened lifespan. These beneficial effects were observed when gene editing was applied before, at and well after the on-set of pathological and behavioral abnormalities. These proof-of-concept data demonstrate that gene editing can be a highly effective therapeutic approach for HD and other inherited neurodegenerative diseases. One Sentence Summary Self-inactivating CRISPR for mutant huntingtin in HD mice achieved long-term rescue of neural pathology, motor deficits, weight loss and survival.

The advent of CRISPR/Cas systems has revolutionized plant genome engineering, transitioning from traditional single-gene edits to sophisticated multiplex genome editing strategies capable of simultaneously targeting multiple loci. This review provides an in-depth examination of CRISPR-mediated multiplexing technologies in plants, emphasizing their molecular mechanisms, delivery systems, and transformative applications in crop improvement. We delineate the evolution of CRISPR systems from early programmable nucleases to diverse Class 2 effectors, including Cas9, Cas12, Cas13, and emerging ultra-compact variants like CasΦ and Cas14. We detail polycistronic gRNA expression platforms—such as tRNA-sgRNA arrays, ribozymes, and Csy4-mediated cleavage—that enable efficient multi-target editing within compact vectors. Furthermore, we explore advanced delivery modalities including Agrobacterium, biolistics, protoplast transfection, and viral vectors, optimized for recalcitrant plant systems. Applications span yield enhancement, disease resistance, abiotic stress tolerance, nutritional fortification, and de novo domestication. Critical challenges including off-target mutagenesis, mosaicism, chromosomal rearrangements, and regulatory constraints are addressed. Finally, we highlight AI-driven sgRNA design, multi-omics integration, and CRISPR libraries as pivotal tools to rationalize and scale multiplex editing. This synthesis underscores multiplex CRISPR as a cornerstone of next-generation plant breeding, with the potential to redefine global agriculture through precision trait stacking and rapid varietal development.

Bacteriophages (phages), the most abundant biological entities on Earth, have long served as both model systems and therapeutic tools. Recent advances in synthetic biology and genetic engineering have revolutionized the capacity to tailor phages with enhanced functionality beyond their natural capabilities. This review outlines the current landscape of synthetic and functional engineering of phages, encompassing both in vivo and in vitro strategies. We describe in vivo approaches such as phage recombineering systems, CRISPR-Cas-assisted editing, and bacterial retron-based methods, as well as synthetic assembly platforms including yeast-based artificial chromosomes, Gibson, Golden Gate, and iPac assemblies. In addition, we explore in vitro rebooting using TXTL (transcription-translation) systems, which offer a flexible alternative to cell-based rebooting but are less effective for large genomes or structurally complex phages. Special focus is given to the design of customized phages for targeted applications, including host range expansion via receptor-binding protein modifications, delivery of antimicrobial proteins or CRISPR payloads, and the construction of bio-contained, non-replicative capsid systems for safe clinical use. Through illustrative examples, we highlight how these technologies enable the transformation of phages into programmable bactericidal agents, precision diagnostics tools, and drug delivery vehicles. Together, these advances establish a powerful foundation for next-generation antimicrobial platforms and synthetic microbiology.