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.

SNX11, a sorting nexin protein localized on the endosomal membrane, is an important protein closely related to protein sorting and endosomal trafficking. Previously, through a genome-wide CRISPR screening, we identified SNX11 as a critical protein for the entry of Dabie bandavirus. SNX11-deletion significantly inhibits the replication of Dabie bandavirus. We further discovered that the loss of SNX11 alters endosomal pH, potentially affecting the release process of Dabie bandavirus from endosomes to the cytoplasm. However, the mechanism by which SNX11 modulates endosomal pH and whether SNX11-deletion similarly inhibits other viruses remain to be elucidated. This study reveals that SNX11 can interact with the V1 subunit of the endosomal proton pump V-ATPase, affecting the expression level of this subunit on the endosomal membrane and thereby disrupting the assembly of V-ATPase. Additionally, we found that SNX11-deletion significantly inhibits the replication of dengue virus, hantavirus, and influenza virus. These findings suggest that SNX11 may be a key protein in the process of viral infection and could serve as a broad-spectrum antiviral target.

Glioblastoma (GBM) is a lethal brain tumor with limited response to standard of care chemoradiotherapy. In this study, we conducted genome-wide CRISPR knockout screening in patient-derived glioblastoma stem cells (GSCs) to identify genetic dependencies of cell survival and therapy resistance. Our screening identified flap endonuclease 1 (FEN1) as a key driver of GSC survival, with enhanced dependency under temozolomide (TMZ) treatment. Genetic perturbation of FEN1 reduced GSC self-renewal and proliferation in vitro, and prolonged survival in a patient-derived xenograft model of GBM. FEN1 inhibition (FEN1i) preferentially affected highly aggressive or recurrent GBM models compared with less aggressive GBMs and healthy neural stem cells. Moreover, FEN1 inhibition synergized with TMZ only in these aggressive FEN1i-sensitive GSCs, providing cancer-selective killing and TMZ sensitization in the most untreatable of GBMs. Mechanistically, FEN1i-sensitive GSCs exhibited greater proliferation and sphere formation, while stalling their proliferation conferred resistance to FEN1 inhibition. Single-cell transcriptomics further linked FEN1 expression to stemness and the DNA damage response, elucidating broader determinants of FEN1 dependency. These findings establish FEN1 as a promising therapeutic target in GBM, offering a strategy for both selective targeting and enhancement of TMZ efficacy in aggressive cancers. Statement of Significance This study identifies FEN1 as a key vulnerability of glioblastoma stem cells, revealing its role in therapy resistance and stemness, and proposes FEN1 inhibition as a strategy to enhance temozolomide efficacy.

RNA editing and maturation are critical regulatory mechanisms in plant organelles, yet their quantification remains technically challenging. Traditional Sanger sequencing lacks sensitivity and reproducibility, whereas advanced next-generation sequencing (NGS) approaches, such as rRNA-depleted RNA-seq or targeted amplicon-seq, involve high costs, complex workflows, and limited accessibility. To address these limitations, we developed a rapid and cost-effective long-read sequencing approach, termed premium PCR sequencing, for digital quantification of RNA-editing and intron retention events in targeted chloroplast transcripts. This method combines multiplexed high-fidelity PCR amplification with Oxford Nanopore sequencing and custom in-house Perl and Python scripts for streamlined data processing, including barcode-based demultiplexing, strand reorientation, alignment to a pseudo-genome, manual editing-site inspection, and splicing variant identification and comparison. Using this platform, we analyzed the ndhB and ndhD transcripts, two chloroplast NAD(P)H dehydrogenase genes with the highest number of known editing sites, in an inducible CRISPR interference (iCRISPRi) system targeting MORF2 , a key RNA-editing factor. Our results revealed MORF2 dosage-dependent reductions in C-to-U editing efficiency, with significant defects observed in the strongly repressed P1-12 line. Moreover, we identified an accumulation of intron-retaining ndhB transcripts, specifically in Dex-treated iCRISPRi lines, indicating impaired chloroplast splicing functions upon MORF2 suppression. The platform achieves single-molecule resolution, robust reproducibility, and high read coverage across biological replicates at a fraction of the cost of lncRNA-seq, making it broadly accessible. This study establishes premium PCR sequencing as a versatile, scalable, and affordable tool for targeted post-transcriptional analysis in plant organelles and expands our understanding of MORF2’s role in chloroplast RNA maturation. Significance Statement We present a rapid, affordable, and reproducible method for digital quantification of RNA editing and intron retention in plant organellar transcripts using nanopore-based long-read sequencing. This platform overcomes key limitations of existing approaches and enables routine, site-specific analysis of post-transcriptional regulation in organelles, including RNA editing and splicing, making it broadly accessible to researchers studying plastid biology, stress responses, and organelle–nucleus communication.

ABSTRACT Background Fibrillins are essential components of the extracellular matrix. Marfan syndrome (MFS), the most common fibrillinopathy, is characterized by severe cardiovascular complications, including cardiac valve abnormalities, myocardial dysfunction, arrhythmias, and, most commonly, thoracic aortic disease. Unfortunately, no definitive medical cure is available. Objectives To establish a zebrafish model of MFS, to enhance understanding of the cardiovascular consequences of fibrillin impairment and identify novel therapeutic targets. Methods CRISPR/Cas9 technology was used to systematically target all zebrafish fibrillin genes. The cardiovascular phenotype was investigated using fluorescent microscopy at embryonic stages and cardiac ultrasound, histology, and synchrotron X-ray imaging in adults. RNA sequencing and drug testing were performed during early development. Results Fibrillin-2b mutant ( fbn2b -/- ) zebrafish had a reproducible phenotype, with a subset of embryos showing endocardial detachment leading to early mortality. Interestingly, the remaining fbn2b -/- zebrafish developed dilation of the bulbus arteriosus, a structure analogous to the aortic root in humans, and survived normally to adulthood. Adult fbn2b -/- zebrafish displayed cardiac valve abnormalities. Transcriptomic analysis of fbn2b -/- embryos suggested the involvement of extracellular matrix remodeling and immune-related pathways. Administration of nebivolol and losartan did not improve the phenotype of fbn2b -/- larvae. Zebrafish lacking fibrillin-1 and/or fibrillin-2a did not show any phenotype. Conclusion Our fbn2b -/- zebrafish model recapitulates key aspects of human cardiovascular manifestations of MFS and can therefore be considered a novel relevant animal model for MFS. Studying this model allows us to broaden the knowledge of the underlying mechanisms of the disease and discover much-needed disease-specific treatment options. CONDENSED ABSTRACT Fibrillin defects lead to severe cardiovascular complications in Marfan syndrome (MFS), including aortic dilation, dissection, and rupture. To model MFS, we generated zebrafish mutants lacking various fibrillin genes. Among these mutant lines, only fibrillin-2b-deficient zebrafish exhibited cardiovascular phenotypes mimicking human disease. Multimodal imaging revealed early cardiac defects, bulbus arteriosus dilation, and valve abnormalities. Transcriptomic analysis identified altered regulation of pathways related to extracellular matrix homeostasis and immune system activation. Compound testing demonstrated the model’s potential for drug discovery. This zebrafish model, recapitulating key cardiovascular features of MFS, provides a valuable platform to investigate disease mechanisms and identify novel treatment strategies.

Abstract γδ T cells are mediators of immunosurveillance used in the clinic. However, their status remains paradoxical. While many display overt traits of adaptive immunity, several major γδ cell subsets including those in barrier tissues make rapid, seemingly TCR-independent responses phenocopying innate lymphoid cells (ILC). While such uncertainty exists, the requirements for γδ T cell-mediated immunosurveillance will remain unclear. This study resolves the paradox, showing that tissue-intrinsic γδ T cells share an absolute real-time dependence on the TCR for their phenotypes, including so-called innate responses to tissue stress and carcinogenesis. While different tissue-intrinsic γδ subsets showed distinct TCR-dependencies, we identified a shared set of TCR-regulated molecules that was likewise disrupted by acute TCR ablation in human γδ T cells. These findings unequivocally distinguish γδ cells from ILC and establish that immunosurveillance by γδ T cells requires an environment conducive to TCR signalling.[139]

ABSTRACT The recent global outbreaks of mpox highlight the urgent need for both fundamental research and antiviral development. However, studying monkeypox virus (MPXV), with its large and complex genome, remains challenging due to the requirement for high-containment facilities. Here, we describe a novel strategy for de novo assembly of MPXV clade IIb genomes in bacterial artificial chromosomes using transformation-associated recombination cloning. Leveraging CRISPR-Cas9 and Lambda Red recombination, we engineered replication-defective MPXV particles with dual deletions of OPG96 ( M2R ) and OPG158 ( A32.5L )—genes essential for virion assembly, that are capable of recapitulating key stages of the viral life cycle. Our work demonstrates the utility of replication-defective MPXV particles as a reliable platform for high-throughput antiviral discovery, offering significant advantages for both fundamental virology studies and therapeutic development against orthopoxviruses.

ABSTRACT Post-transcriptional modifications expand the information encoded by an mRNA. These dynamic and reversible modifications are specifically recognized by reader RNA-binding proteins (RBPs), which mediate the regulation of gene expression, RNA processing, localization, stability, and translation. Given their crucial functions, any disruptions in the normal activity of these readers can have significant implications for cellular health. Consequently, the dysregulation of these RBPs has been associated with neurodegenerative disorders, cancers, and viral infections. Therefore, there has been growing interest in targeting reader RBPs as a potential therapeutic strategy since developing molecules that restore proper RNA processing and function may offer a promising avenue for treating diseases. In this work, we coupled our previously established live-cell RNA-protein interaction (RPI) assay, RNA interaction with Protein-mediated Complementation Assay (RiPCA), with CRISPR technology to build a new platform, CRISPR RiPCA. As a model for development, we utilized the interaction of eukaryotic translation initiation factor 4E (eIF4E), a reader RBP that binds to the m 7 GpppX cap present at the 5′ terminus of coding mRNAs, with an m 7 G capped RNA substrate. Using eIF4E CRISPR RiPCA, we demonstrate our technology’s potential for measuring on-target activity of inhibitors of the eIF4E RPI of relevance to cancer drug discovery.

ABSTRACT LMNA -related congenital muscular dystrophy (L-CMD) is amongst the most severe forms of laminopathies, which are diseases caused by pathogenic variants in the LMNA gene. LMNA encodes the proteins LAMINs A and C, which assemble with LAMIN B1 and B2 to form the nuclear lamina: a meshwork providing structural stability to the nucleus that also regulates chromatin organisation and gene expression. Research into L-CMD mechanisms and therapies i hindered by lack of humanised, tissue-specific models that accurately recapitulate disease phenotypes. We previously reported that LMNA -mutant induced pluripotent stem cell (iPSC)-derived skeletal muscle cells have nuclear shape abnormalities and LAMIN A/C protein mislocalisation. Here, we expand the selection of L-CMD patient-derived iPSCs and validate disease-associated readouts using a transgene-free based protocol which more accurately mimics skeletal myogenesis. Results showed no defects in developmental myogenesis, but recapitulation of pathological nuclear shape abnormalities in 2D and 3D cultures, nuclear envelope protein mislocalisation and transcriptomic alterations across multiple pathogenic LMNA variants. We then utilised this platform to assess LMNA gene editing strategies. CRISPR-based exon removal generated stable RNA and protein LAMIN A/C species, without significant normalisation of nuclear morphological phenotypes. Conversely, precise editing of the same mutation showed complete reversal of disease-associated nuclear morphometrics. Our data provide the foundation for a humanised in vitro disease and therapy modelling platform for this complex and severe muscle disorder. GRAPHICAL ABSTRACT (created with BioRender.com) HIGHLIGHTS LMNA -mutant iPSCs undergo efficient skeletal myogenesis upon transgene-free, small molecule-based lineage-directed differentiation. LMNA -mutant iPSCs recapitulate hallmark disease-associated nuclear phenotypes and show a pro-inflammatory transcriptional profile LMNA -mutant iPSC-derived muscle cells enable testing of genetic therapies CRISPR-edited L-CMD iPSC-derived myogenic cells show amelioration of disease-associated phenotypic readouts

Variation in leaf complexity modulates light capture and is a target for crop enhancement. Soybean typically has compound leaves with three leaflets each, but a spontaneous mutation, designated lf2, possesses seven leaflets, offering a means to dissect the molecular mechanisms specifying leaflet number and assess its potential for soybean improvement. However, the developmental and genetic bases of the lf2 mutation remain unknown. Here, we characterize the seven-leaflet phenotype and identify the mutation responsible for the phenotypic changes. Microscopic examination of leaf emergence sites revealed that the seven-leaflet phenotype arises in a two-step process: five leaflets form initially followed by secondary leaflet initiation at the margins of the central leaflet. Genetic mapping delineated lf2 to a ∼2.5 Mb region at the start of chromosome 11. Fortuitously, integration of pedigree analysis with comparative analysis of genomic sequences from the region pinpointed a 2-bp deletion in the coding sequence of a gene, which is homologous to the Arabidopsis KNAT7 encoding a KNOTTED1-LIKE HOMEOBOX 2 transcription factor, as the sole candidate for Lf2. The deletion is predicted to result in disruption of the putative DNA-binding homeodomain. Expression of the wild-type allele of the candidate gene in the seven-leaflet lf2 mutant restored the three-leaflet phenotype, while disruption of the wild-type allele through CRISPR-Cas9 editing induced extra leaflet formation. This study advances our understanding of leaflet formation in legumes and provides a template for utilizing compound leaf architecture to optimize photosynthetic efficiency and yield in soybean.

ABSTRACT Invasive lobular carcinoma (ILC) is a common subtype of breast cancer that is defined in part by genetic loss of CDH1 caused by mutation or deletion, leading to loss of cell adhesion protein E-cadherin in >90% of ILC. Genetic loss of CDH1 is an early event in ILC oncogenesis, yet the mechanisms by which CDH1/ E-cadherin acts as a tumor suppressor are not well understood. To study how early CDH1 loss drives ILC oncogenesis, we used a series of non-transformed human mammary epithelial cell (HMEC) models to target CDH1 /E-cadherin, inhibiting extracellular E-cadherin signaling using antibodies versus modeling genetic CDH1 loss using siRNA or knockout via CRISPR/Cas9. Through transcriptome analyses across four HMEC models, we found that the mode of E-cadherin loss or suppression is critical for the subsequent phenotype. Antibody-mediated inhibition of cell-cell contacts induced gene signatures of epithelial-mesenchymal transition (EMT), consistent with the role of E-cadherin suppression during the EMT process. Conversely, genetic CDH1 loss – as in ILC oncogenesis – repressed EMT signatures, and instead remodeled gene expression toward a luminal epithelial phenotype. Using single cell transcriptomics and flow cytometry analyses of cell lineage markers, we found that genetic loss of CDH1 reprogrammed cells to a luminal progenitor-like phenotype. By isolating luminal versus basal cells prior to CDH1 knockout, we found that CDH1 loss led to remodeling of lineage identity in both populations, converging on a new lineage homeostasis with a luminal progenitor-like phenotype. Consistent with increased progenitor features, CDH1 loss enhanced proliferative capacity over the finite lifespan of the HMECs, highlighting a feature of early CDH1 loss that may contribute to clonal advantage during tumor initiation. Our findings support that inhibition of E-cadherin results in different transcriptional response compared to CDH1 loss, with the latter driving a transcriptional and phenotypic state characteristic of a luminal progenitor-like population, which offers new insight into early events in ILC oncogenesis.

Intercellular communication is essential for distributed genetic circuits operating across cells in multicellular consortia. While diverse signalling molecules have been employed--ranging from quorum sensing signals, secondary metabolites, and pheromones to peptides, and nucleic acids--phage-packaged DNA offers a highly programmable method for communicating information between cells. Here, we present a library of five M13 phagemid variants with distinct replication origins, including those based on the Standard European Vector Architecture (SEVA) family, designed to tune the growth and secretion dynamics of sender strains. We systematically characterize how intracellular phagemid copy number varies with cellular growth physiology and how this, in turn, affects phage secretion rates. In co-cultures, these dynamics influence resource competition and modulate communication outcomes between sender and receiver cells. Leveraging the intercellular CRISPR interference (i-CRISPRi) system, we quantify phagemid transfer frequencies and identify rapid-transfer variants that enable efficient, low-burden communication. The phagemid toolbox developed here expands the repertoire of available phagemids for DNA-payload delivery applications and for implementing intercellular communication in multicellular circuits.

Pediatric cancers pose significant treatment challenges due to their biological heterogeneity and variable responses to chemotherapy. SLFN11, a DNA/RNA helicase-like protein known to sensitize adult tumors to DNA-damaging agents, remains underexplored in pediatric malignancies. Here, we investigate the role of SLFN11 across pediatric Wilms tumor, osteosarcoma, and medulloblastoma using integrated bioinformatics, epigenetic profiling, and functional assays. In silico analysis of TARGET and ICGC datasets revealed distinct correlations between SLFN11 expression and patient survival, with positive, negative, or neutral predictive value depending on tumor type. Baseline expression and promoter methylation analysis in pediatric cancer cell lines demonstrated epigenetic regulation of SLFN11, similar to adult cancers. Using CRISPR-dCas9-mediated activation, we successfully upregulated SLFN11, which significantly enhanced sensitivity to cisplatin and the PARP inhibitor talazoparib across all tested cell lines. Transcriptomic profiling under cisplatin treatment indicated that SLFN11 modulates DNA damage response and MAPK signaling pathways, potentially contributing to chemotherapy sensitivity. These findings establish SLFN11 as a context-dependent predictive biomarker and a potential therapeutic target to overcome chemoresistance in pediatric solid cancers.

Streptococcus agalactiae (group B Streptococcus ; GBS) is a leading cause of neonatal sepsis and meningitis. Despite advances in molecular microbiology, GBS genome engineering remains laborious due to inefficient mutagenesis protocols. Here, we report a versatile and rapid Cas12a-based toolkit for GBS genetic manipulation. We developed two shuttle plasmids—pGBSedit for genome editing and pGBScrispri for inducible CRISPR interference—derived from an Enterococcus faecium system and optimized for GBS. Using these tools, we achieved targeted gene insertions, markerless deletions, and efficient, template-free mutagenesis via alternative end-joining repair. Furthermore, a catalytically inactive dCas12a variant enabled inducible gene silencing, with strand-specific targeting effects. The system demonstrated broad applicability across multiple GBS strains and minimal off-target activity, as confirmed by whole-genome sequencing. This Cas12a-based platform offers a rapid, flexible, and scalable approach to genetic studies in GBS, facilitating functional genomics and accelerating pathogenesis research.

Gliomas are the most common primary intracranial tumors, comprising 81% of malignant brain tumors, and currently lack effective therapies. Recent advances in molecular biology have shown that cancer cells exploit microtubule-associated proteins (MAPs) under stress to activate various signaling pathways. This study investigates the role of Doublecortin (DCX) in glioma metabolism and its impact on tumor proliferation. In this study, CRISPR-engineered glioma models with DCX overexpression or knockdown were analyzed using integrated genomic, transcriptomic, and metabolomic approaches. Metabolic activity was assessed via RNA sequencing, Seahorse assays, and targeted mass spectrometry. Pharmacological inhibition of key pathways validated functional dependencies. We demonstrate that gliomas enriched with DCX exhibit elevated glycolytic activity while also relying on cellular respiration and oxidative phosphorylation (OXPHOS) for energy to support the abnormal proliferation of glioma cells. Upon integrative analysis of enriched genes and proteins, we observed genetic and metabolome-level signatures associated with differences in central carbon and energy metabolism in CRISPR-modified glioma cells expressing high DCX. Whole-genome transcriptome analysis revealed enriched metabolic entities promoting hydrolysis of glutamine and glutaminolysis in glioma cells and inhibition of selected differentially enriched genes with small molecule inhibitors abrogated metabolic enrichment and resulted in reduced energy levels and protein translation required for aberrant growth. Finally, we establish that DCX stimulates glutaminolysis to regulate homeostasis for energy supplements in glioma cells. Targeting DCX-mediated metabolic pathways may provide a novel therapeutic approach for glioblastoma, highlighting the potential for innovative treatments in this challenging disease.

CRISPR-Cas systems provide adaptive immunity in bacteria and archaea against mobile genetic elements, but the role they play in gene exchange and speciation remains unclear. Here, we investigated how CRISPR-Cas targeting affects mating and gene exchange in the halophilic archaeon Haloferax volcanii . Surprisingly, we found that CRISPR-Cas targeting significantly increased mating efficiency between members of the same species, in contrast to its previously documented role in reducing inter-species mating. This enhanced mating efficiency was dependent on the Cas3 nuclease/helicase and extended beyond the targeted genomic regions. Further analysis revealed that CRISPR-Cas targeting promoted biased recombination in favour of the targeting strain during mating, resulting in an increased proportion of recombinant progeny that are positive for CRISPR-Cas. To test whether an increase in recombination is sufficient to increase mating efficiency, we tested whether strains lacking the MRE11-RAD50 complex, which are known to have elevated recombination activity, also exhibited higher mating success. Indeed, these strains showed higher mating, as did cells that were exposed to DNA damage using methyl methanesulfonate. These findings suggest that CRISPR-Cas systems may contribute to speciation by facilitating within-species gene exchange while limiting between-species genetic transfer, thereby maintaining species boundaries.

Efficient, safe, and cell-selective intracellular delivery remains a bottleneck for scalable and cost-effective manufacturing of cell therapies. Here, we introduce Selective Permeabilization using Impedance Cytometry (SPICy) that couples multifrequency single-cell impedance cytometry with real-time, feedback-controlled, low-voltage single-cell electroporation. Electric field focusing in a 3-D printed biconical micro-aperture confines both sensing and electroporation to a microscale zone, enabling continuous-flow operation and the use of low voltages ( 80 %) and high (>90 %) cell viability. Delivery of a range of different cargo sizes (4–500 kDa), GFP mRNA expression, CRISPR-Cas9 based knock-out and delivery to a variety of different cell lines, primary human T cells and peripheral blood mononuclear cells (PBMCs) was also demonstrated. Using heterogenous or mixed samples, selective delivery to both cell lines, and primary immune cell subpopulations, from PBMCs, was demonstrated. SPICy thus provides a label-free, continuous flow, targeted non-viral platform for precision cell engineering.

Mitochondria contribute to compartmentalized metabolism in eukaryotic cells, supporting key enzymatic reactions for cell function and energy homeostasis. However, this compartmentalization necessitates regulated metabolite transport across mitochondrial membranes. Although many transport proteins have been identified, several mitochondrial amino acid transporters remain largely uncharacterized. Using CRISPR-Cas9–mediated candidate transporter knockouts coupled with assessment of metabolite transport via a mitochondrial swelling assay, we identify SFXN1, previously characterized for its role in mitochondrial serine transport, as a protein that mediates mitochondrial transport of a range of other polar neutral amino acids including proline, glycine, threonine, taurine, hypotaurine, β-alanine, and γ-aminobutyric acid (GABA). Furthermore, the SFXN1 paralogues SFXN2 and SFXN3 partially complement loss of SFXN1 to enable glycine transport, while SFXN2 and SFXN5 partially complement loss of SFXN1 to enable GABA transport. Altogether, these data suggest that sideroflexins facilitate the transport of polar neutral amino acids across the inner mitochondrial membrane.

RNA viruses pose significant threats to global health, causing diseases such as COVID-19, HIV/AIDS, influenza, and dengue. These viruses are characterized by high mutation rates, rapid evolution, and the ability to evade traditional antiviral therapies, making effective treatment and prevention particularly challenging. In recent years, CRISPR/Cas13 has emerged as a promising antiviral tool due to its ability to specifically target and degrade viral RNA. Unlike conventional antiviral strategies, Cas13 functions at the RNA level, providing a broad-spectrum and programmable approach to combating RNA viruses. Its flexibility allows for rapid adaptation of guide RNAs to counteract emerging viral variants, making it particularly suitable for highly diverse viruses such as SARS-CoV-2 and HIV. This review discusses up-to-date applications of CRISPR/Cas13 in targeting a wide range of RNA viruses, including SARS-CoV-2, HIV, Dengue, Influenza and other RNA viruses, with the main focus on its potential in therapeutic context. Preclinical studies have demonstrated Cas13’s efficacy in degrading viral RNA and inhibiting replication, with applications spanning prophylactic interventions to post-infection treatments. However, challenges such as collateral cleavage, inefficient delivery, potential immunogenicity as well as development of a concurrent ethical basis must be addressed before clinical translation. Future research should focus on optimizing crRNA design, improving delivery systems, and conducting rigorous preclinical evaluations to enhance specificity, safety, and therapeutic efficacy. With continued advancements, CRISPR/Cas13 holds great promise as a revolutionary antiviral strategy, offering new solutions to combat some of the world’s most persistent viral threats.

Abstract Wildlife crime—including illegal poaching, breeding, and trafficking—is the second most widespread form of transnational crime globally, generating over USD $20 billion annually. These illicit activities not only drive numerous species toward extinction but also destabilize ecosystems, threaten public health through zoonotic spillovers and disrupt the socio-economic stability of communities that rely on biodiversity. The growing scale and complexity of these crimes demand advanced forensic approaches that can deliver robust, legally admissible evidence. With the advent of the genomic era, molecular technologies have revolutionized wildlife forensics by enabling precise species and sex identification, individual assignment, and population tracking. This review explores the integration of genomic tools—including DNA barcoding, short tandem repeats (STRs), single nucleotide polymorphisms (SNPs), whole genome sequencing (WGS), and environmental DNA (eDNA) approaches—into wildlife crime investigations. Special focus is given to cutting-edge techniques such as CRISPR-based species detection, metagenomics for tracking the illegal wildlife trade, and the deployment of portable sequencers for in-field genotyping. The review also addresses pressing challenges and limitations such as the ethical use of genetic data, concerns about biopiracy, and the complexities of integrating genomic evidence into judicial and conservation frameworks. By analyzing the technological, ethical, and policy dimensions of genomics in wildlife forensics, this paper aims to guide researchers, law enforcement agencies, and policymakers toward a cohesive strategy for combating poaching and illegal wildlife trade in the genomic era.

Summary Neurological diseases (NDs) are a major source of unmet medical need, and translational insights have been hampered by complex underlying pathophysiologies and limitations of experimental models. Noncoding single nucleotide polymorphisms (SNPs) at hundreds of loci have been linked to ND risk by genome-wide association studies (GWAS), but the causal genes and pathways are largely unknown. Despite the multicellular pathology of complex traits like multiple sclerosis (MS), functional studies that aim to characterize the molecular impact of disease-associated SNPs often investigate all SNPs linked to disease in the same cellular context. Here, we combine a computational approach to predict the pathogenic cell type of individual risk loci with functional CRISPR perturbation studies in iPSC-derived microglia cells (iMGLs). ND SNP enrichment in cell type-specific enhancers is similar between primary and iPSC-derived cells, and mechanistically supported by shared enhancer-promoter interactions. We apply a novel Perturb-seq platform to interrogate MS risk SNPs in iMGL, identifying likely cis -acting causal risk genes at 5 of 9 loci, as well as downstream differentially expressed genes (DEGs). Despite being found in trans to MS risk SNPs, downstream DEGs are substantially enriched for MS heritability. Downstream DEGs from all 5 target genes show significant overlap, converging on genes related to cytokinesis, phagocytosis, and mitochondrial metabolism. We then compared downstream DEGs to gene expression patterns observed in MS patient tissue studies and observed marked similarities, demonstrating that genes dysregulated as the result of GWAS loci perturbation mirrored effects observed in microglia found in MS patient lesions. Collectively, these results demonstrate that cell type aware functional studies can be used to translate ND SNP associations into mechanistic insights and reveal novel convergent biological mechanisms underlying complex traits.

SUMMARY The von Hippel-Lindau tumor suppressor (VHL) is a component of a ubiquitin ligase complex that normally controls cellular responses to hypoxia. Endogenous VHL is also utilized by proteolysis-targeting chimera (PROTAC) protein degraders, a promising class of anti-cancer agents. VHL is broadly essential for cell proliferation, yet it is a key tumor suppressor in renal cell carcinoma. To understand the functional consequences of VHL loss, and to identify targeted approaches for the elimination of VHL null cells, we have used genome-wide CRISPR-Cas9 screening in human renal epithelial cells. We find that, upon VHL loss, the HIF1A/ARNT complex is the central inhibitor of cellular fitness, suppressing mitochondrial respiration, and that VHL null cells show HIF1A-dependent molecular vulnerabilities that can be targeted pharmacologically. Combined VHL/HIF1A inactivation in breast and esophageal cancer cells can also provide resistance to ARV-771, a VHL-based bromodomain degrader that has anti-cancer activity. HIF1A stabilization can thus provide opportunities for early intervention in neoplastic VHL clones, and the VHL-HIF1A axis may be relevant for the development of resistance to the emerging class of PROTAC-based cancer therapies.

Abstract Nucleoredoxin (NRX), a member of the thioredoxin (TRX) superfamily, is involved in regulating plant growth and development, as well as abiotic and biotic stress processes, but it is not clear whether NRX participates in regulating the drought resistance of foxtail millet. In this study, the SiNRX1 of foxtail millet was knocked out using CRISPR/Cas9 system, and the drought resistance of sinrx1 mutants was identified at both the germination stage and the seedling stage. Moreover, through transcriptome sequencing and data-independent acquisition (DIA) quantitative proteomics determination of sinrx1 mutants and wild types (WT) at the seedling stage under drought and control conditions, the molecular mechanism of SiNRX1 regulating drought resistance was preliminarily analyzed. The results indicated that after 7 days of simulated drought treatment during the germination period, the germination rate, root length and bud length of the sinrx1 mutant decreased significantly in comparison with the WT. During the seedling stage under drought stress, the survival rate, chlorophyll content, proline content, peroxidase (POD) activity and catalase (CAT) activity of the sinrx1 mutant decreased markedly compared to those of the WT plants. However, the malondialdehyde (MDA) content of the sinrx1 mutant was significantly higher than that of the WT. This indicates that, during the germination and seedling stages, the drought resistance of the sinrx1 mutant decreased significantly compared to that of the WT. The transcriptome findings suggested that the 2253 differentially expressed genes (DEGs) (1179 up-regulated DEGs and 1074 down-regulated DEGs) are drought-responsive genes specifically influenced by SiNRX1 . The proteomic results suggests that the 155 differentially expressed proteins (DEPs) (127 up-regulated DEPs and 28 down-regulated DEPs) are drought-responsive proteins specifically influenced by SiNRX1. Moreover, 2253 DEGs and 155 DEPs were significantly and jointly enriched in the phenylpropanoid biosynthesis pathway. This study offers theoretical guidance for the analysis of the drought resistance mechanism of foxtail millet plants and for drought resistance breeding.