Search Results (219)

Prime editing (PE) has emerged as a transformative genome editing technology, enabling precise base substitutions, insertions, and deletions without inducing double-strand DNA breaks (DSBs). However, its application in zebrafish remains limited by low efficiency. Here, we leveraged PE7, a state-of-the-art PE system, combined with La-accessible prime editing guide RNAs (pegRNAs), to enhance editing efficiency in zebrafish. By co-incubating PE7 protein with La-accessible pegRNAs to form ribonucleoprotein (RNP) complexes and microinjecting these complexes into zebrafish embryos, we achieved up to 15.99% editing efficiency at target loci—an improvement of 6.81- to 11.46-fold over PE2. Additionally, we observed 16.60% 6 bp insertions and 13.18% 10 bp deletions at the adgrf3b locus, representing a 3.13-fold increase over PE2. Finally, we used PE to introduce desired edits at the tyr locus, successfully generating zebrafish with the tyr P302L mutation that exhibited melanin reduction. These findings demonstrate that PE7 significantly enhances prime editing efficiency in fish, providing novel tools for functional gene studies and genetic breeding in aquatic species. Full article

The recent discovery of Orthonairovirus songlingense (SGLV) and Norwavirus beijiense (BJNV) in China has raised significant concern due to their potential to cause severe human disease. However, little is known about the structural features and function of their nucleoproteins, which play a key role in the viral life cycle. By combining small-angle X-ray scattering (SAXS) data and AlphaFold 3 simulations, we reconstructed the BJNV and SGLV nucleoprotein structures for the first time. The SGLV and BJNV nucleoproteins have structures that are broadly similar to those of Orthonairovirus haemorrhagiae (CCHFV) nucleoproteins despite low sequence similarity. Based on structural analysis, several residues located in the positively charged region of BJNV and SGLV nucleoproteins have been indicated to be important for viral RNA binding. A positively charged RNA-binding crevice runs along the interior of the SGLV and BJNV ribonucleoprotein complex (RNP), shielding the viral RNA. Despite the high structural similarity between SGLV and BJNV nucleoprotein monomers, their RNPs adopt distinct conformations. These findings provide important insights into the molecular mechanisms of viral genome packaging and replication in these emerging pathogens. Also, our work demonstrates that experimental SAXS data can validate and improve predicted AlphaFold 3 structures to reflect their solution structure and also provides the first low-resolution structures of the BJNV and SGLV nucleoproteins for the future development of POC tests, vaccines, and antiviral drugs. Full article

Stress granule formation is a type of liquid–liquid phase separation in the cytoplasm, leading to RNA–protein condensates that are associated with various cellular stress responses and implicated in numerous pathologies, including cancer, neurodegeneration, inflammation, and cellular senescence. One of the key components of mammalian stress granules is the DEAD-box RNA helicase DDX3, which unwinds RNA in an ATP-dependent manner. DDX3 is involved in multiple steps of RNA metabolism, facilitating gene transcription, splicing, and nuclear export and regulating cytoplasmic translation. In this study, we investigate the role of the RNA helicase DDX3’s enzymatic activity in shaping the RNA content of ribonucleoprotein (RNP) condensates formed during arsenite-induced stress by inhibiting DDX3 activity with RK-33, a small molecule previously shown to be effective in cancer clinical studies. Using the human osteosarcoma U2OS cell line, we purified the RNP granule fraction and performed RNA sequencing to assess changes in the RNA pool. Our results reveal that RK-33 treatment alters the composition of non-coding RNAs within the RNP granule fraction. We observed a DDX3-dependent increase in circular RNA (circRNA) content and alterations in the granule-associated intronic RNAs, suggesting a novel role for DDX3 in regulating the cytoplasmic redistribution of non-coding RNAs. Full article

The CRISPR/Cas9 system is a powerful genome-editing tool that is applied in baculovirus engineering. In this study, we present the first report of the AcMNPV genome deletions for bioproduction purposes, using a dual single-guide RNA (sgRNA) CRISPR/Cas9 approach. We used this method to remove nonessential genes for the budded virus and boost recombinant protein yields when applied as BEVS. We show that the co-delivery of two distinct ribonucleoprotein (RNP) complexes, each assembled with a sgRNA and Cas9, into Sf9 insect cells efficiently generated deletions of fragments containing tandem genes in the genome. To evaluate the potential of this method, we assessed the expression of two model proteins, eGFP and HRPc, in insect cells and larvae. The gene deletions had diverse effects on protein expression: some significantly enhanced it while others reduced production. These results indicate that, although the targeted genes are nonessential, their removal can differentially affect recombinant protein yields depending on the host. Notably, HRPC expression increased up to 3.1-fold in Spodoptera frugiperda larvae. These findings validate an effective strategy for developing minimized baculovirus genomes and demonstrate that dual-guide CRISPR/Cas9 editing is a rapid and precise tool for baculovirus genome engineering. Full article

The virus-first theory presents a model in which viral lineages emerged before cells. This proposal aims to give the theory greater relevance by offering a plausible evolutionary framework that explains both (i) the origin of viruses from prebiotic chemistry and (ii) how viruses contributed to the emergence of cells. Here, we propose that viruses should be understood as a distinct class of ribonucleoprotein (RNP) systems, some of which evolved directly from the RNP-world. In our model, simple progenotes produced capsid-like particles through the evolution of a single gene encoding a self-assembling peptide. This allowed the formation of icosahedral shells around RNA genomes, as observed today in certain viral families whose capsids consist of ~60 identical subunits derived from a single gene product. These early capsids enabled mobility and protection, representing key intermediates toward biological complexity. Over time, some of those populations acquired additional peptides and evolved more elaborate architectures. Finally, the incorporation of lipid-binding domains in those capsid-like peptides allowed the formation of proteolipidic membranes akin to those found in modern cells. This model provides a gradualistic and logically coherent evolutionary path from the RNP-world to the emergence of cellular life, emphasizing the foundational role of viruses in early evolution. Full article

While the U1 small nuclear ribonucleoprotein (snRNP) plays a crucial role in early spliceosome assembly, the mechanisms by which it coordinates with other splicing factors for efficient assembly remain elusive. This study aimed to examine the role of the Swt21 protein in regulating U1 snRNP in Saccharomyces cerevisiae. Swt21p was required for efficient pre-mRNA splicing both in vivo and in vitro. Deletion of SWT21 altered the splicing patterns of two-intron SUS1 RNA, causing intron retention and exon skipping. Spliceosome assembly analysis revealed that in the pre-B complex, the levels of U1 protein components, as well as U1 RNA, were decreased following SWT21 deletion, highlighting the compromised stability of U1 snRNP during this stage. Consistently, in the absence of Swt21p, free isoform of U1 component Nam8p was observed, and its proper nuclear localization was disrupted, demonstrating the functional importance of Swp21p for the stable association of Nam8p with U1 snRNP. Moreover, Swt21p remained primarily in a free state under physiological conditions and did not associate with the pre-B complex. Additionally, TAP analysis revealed that Swt21p-associated proteins are involved in cellular processes beyond splicing. These findings collectively indicate that Swt21p functions as a spliceosome regulator rather than a core component and support a model wherein Swt21p contributes to U1 snRNP stability during early spliceosome assembly. Full article

Alternative splicing (AS) is a pivotal post-transcriptional mechanism that expands the functional diversity of the proteome by enabling a single gene to generate multiple mRNA and protein isoforms. This process, which involves the differential inclusion or exclusion of exons and introns, is tightly regulated by splicing factors (SFs), such as serine/arginine-rich proteins (SRs), heterogeneous nuclear ribonucleoproteins (hnRNPs), and RNA-binding motif (RBM) proteins. These factors recognize specific sequences, including 5′ and 3′ splice sites and branch points, to ensure precise splicing. While AS is essential for normal cellular function, its dysregulation is increasingly implicated in cancer pathogenesis. Aberrant splicing can lead to the production of oncogenic isoforms that promote tumorigenesis, metastasis, and resistance to therapy. Furthermore, such abnormalities can cause the loss of tumor-suppressing activity, thereby contributing to cancer development. Importantly, abnormal AS events can generate neoantigens, which are presented on tumor cell surfaces via major histocompatibility complex (MHC) molecules, suggesting novel targets for cancer immunotherapy. Additionally, splice-switching oligonucleotides (SSOs) have shown promise as therapeutic agents because they modulate splicing patterns to restore normal gene function or induce tumor-suppressive isoforms. This review explores the mechanisms of AS dysregulation in cancer, its role in tumor progression, and its potential as a therapeutic target. We also discuss innovative technologies, such as high-throughput sequencing and computational approaches, that are revolutionizing the study of AS in cancer. Finally, we address the challenges and future prospects of targeting AS for personalized cancer therapies, emphasizing its potential in precision medicine. Full article

Hepatocyte transplantation (HTx) combined with ex vivo gene therapy has garnered significant interest due to its potential for treating many inherited metabolic liver diseases. The biggest obstacle for HTx is achieving sufficient engraftment levels to rescue diseased phenotypes, which becomes more challenging when combined with ex vivo gene editing techniques. However, recent technological advancements have improved electroporation delivery efficiency, cell viability, and scalability for cell therapy. We recently demonstrated the impacts of electroporation for cell-based gene therapy in a mouse model of hereditary tyrosinemia type 1 (HT1). Here, we explore the use of the clinical-grade electroporator, the MaxCyte ExPERT GTx, utilized in the first FDA-approved CRISPR therapy, Casgevy, and evaluate its potential in primary hepatocytes in terms of delivery efficiency and cell viability. We assessed the gene editing efficiency and post-transplantation engraftment of hepatocytes from mTmG mice electroporated with CRISPR-Cas9-ribonucleoproteins (RNPs) targeting 4-hydroxyphenylpyruvate dioxygenase (Hpd) in a fumarylacetoacetate hydrolase (Fah)-deficient mouse model of HT1. After surgery, Fah^-/- graft recipients were cycled off and on nitisinone to achieve independence from drug-induced Hpd inhibition, an indicator of HT1 disease correction. Transplanted hepatocytes subjected to electroporation using the GTx system had a cell viability of 89.9% and 100% on-target gene editing efficiency. Recipients transplanted with GTx-electroporated cells showed a smaller weight reduction than controls transplanted with untransfected cells (7.9% and 13.8%, respectively). Further, there were no mortalities in the GTx-recipient mice, whereas there was 25% mortality in the control recipients. Mean donor cell engraftment was significantly higher in GTx-recipient mice compared to untransfected control recipients (97.9% and 81.6%, respectively). Our results indicate that the GTx system does not negatively impact hepatocyte functionality and engraftment potential, thereby demonstrating the promise of GTx electroporation in hepatocytes as a viable cell therapy for treating genetic diseases that affect the liver. Full article

Agasicles hygrophila is the most effective natural enemy for the control of the invasive weed Alternanthera philoxeroides (Mart.) Griseb. However, research on the gene function and potential genetic improvement of A. hygrophila is limited due to a lack of effective genetic tools. In this study, we employed the A. hygrophila white (AhW) gene as a target gene to develop a CRISPR/Cas9-based gene editing method applicable to A. hygrophila. We showed that injection of Cas9/sgRNA ribonucleoprotein complexes (RNPs) of the AhW gene into pre-blastoderm eggs induced genetic insertion and deletion mutations, leading to white eyes. Our results demonstrate that CRISPR/Cas9-mediated gene editing is possible in A. hygrophila, offering a valuable tool for studies of functional genomics and genetic improvement of A. hygrophila, which could potentially lead to more effective control of invasive weeds through the development of improved strains of this biocontrol agent. In addition, the white-eyed mutant strain we developed could potentially be useful for other transgenic research studies on this species. Full article

The transcription and replication of the genome of influenza A virus (IAV) take place in the nucleus of infected cells, which is catalyzed by the viral ribonucleoprotein (vRNP) complex. The nuclear import of the vRNP complex and its component proteins is essential for the efficient replication of IAV and is therefore prone to be targeted by host restriction factors. Herein, we found that host cellular protein keratin 6A (KRT6A) is a negative regulator of IAV replication because siRNA-mediated knockdown of KRT6A expression increased the growth titers of IAV, whereas exogenous overexpression of KRT6A reduced viral yields. The nuclear import of incoming vRNP complexes and newly synthesized nucleoprotein (NP) was significantly impaired when KRT6A was overexpressed. Further studies showed that KRT6A interacts with the four vRNP complex proteins—polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and NP. Notably, the interaction between KRT6A and vRNP complex proteins had no effect on the nuclear import of PB2 or the PB1-PA heterodimer but impaired the interaction between NP and the nuclear import adaptor importin α3, thereby inhibiting the nuclear import of incoming vRNP complexes and newly synthesized NP. Moreover, KRT6A was further shown to suppress the assembly of the vRNP complex and consequently reduce viral polymerase activity. Together, our data uncover a novel role of KRT6A in counteracting the nuclear import and functions of the vRNP complex, thereby restricting the replication of IAV. Full article

Background/Objectives: N-Myristoyltransferase inhibitors (NMTi) represent a novel antiviral strategy against mammarenaviruses such as Lassa and Junin viruses. The Z matrix protein inhibits viral ribonucleoprotein (vRNP) activity in a dose-dependent manner. Here, we investigated whether Z-mediated vRNP inhibition depends on Z myristoylation or oligomerization. Methods: We used HEK293T cells transfected with wild-type (WT) or G2A-mutated Z constructs in LCMV minigenome (MG) assays. Cells were treated with the NMTi IMP-1088 and the proteasome inhibitor MG132. Z protein expression, vRNP activity, and VLP production were analyzed by immunofluorescence, western blotting, and colocalization analyses. Results: IMP-1088 treatment led to proteasome-mediated degradation of Z, reducing its inhibition of vRNP activity, which was restored by MG132. The non-myristoylated Z G2A mutant retained vRNP inhibitory activity but showed impaired oligomerization and budding capacity. These findings demonstrate that Z-mediated vRNP inhibition is independent of myristoylation and oligomerization. Conclusions: Z myristoylation and oligomerization are not required for its inhibitory vRNP activity. Targeting Z myristoylation with NMTi impairs virus assembly and budding without affecting Z-mediated inhibition of vRNP activity, supporting the development of NMTi as a promising broad-spectrum antiviral strategy against mammarenaviruses. Full article

Seasonal influenza epidemics caused by influenza A viruses (IAV) and influenza B viruses (IBV) pose a substantial public health burden. Despite the significant impact of IBV, its restricted host range and the absence of documented pandemics have resulted in limited research attention relative to IAV. Understanding the viral infection mechanisms of both IAV and IBV is crucial for controlling seasonal epidemics. Previously, we demonstrated that 3′-O-sulfated galactosylceramide sulfatide binds to IAV and enhances viral replication, a finding with potential therapeutic implications. However, the role sulfatide plays in other influenza virus infections, including those caused by IBV, remains unknown. Accordingly, in this paper, we investigate the function of sulfatide during IBV infection. We demonstrate that sulfatide binds to IBV hemagglutinin (HA), and that sulfatide overexpression significantly enhances IBV replication, whereas treatment with sulfatase or an anti-sulfatide antibody markedly suppressed IBV replication. Moreover, further tests involving the inhibition of sulfatide biosynthesis resulted in the suppression of viral replication with impaired nuclear export of viral ribonucleoproteins (vRNPs). These findings establish that sulfatide is a critical regulator of IBV replication, which parallels its role in IAV infection, and suggest that targeting sulfatide-virus interactions can lead to broad-spectrum therapeutic strategies against influenza virus. Full article

Climate change profoundly impacts the health, productivity, and resilience of forest ecosystems and threatens the sustainability of forest products and wood-based industries. Innovations to enhance tree growth, development, and adaptation offer unprecedented opportunities to strengthen ecosystem resilience and mitigate the effects of climate change. Here, we established a method for protoplast isolation, purification, and CRISPR-Cas ribonucleoprotein (RNP) delivery in Pinus taeda and Abies fraseri as a step towards accelerating the genetic improvement of these coniferous tree species. In this system, purified protoplasts could be isolated from somatic embryos with up to 2 × 10⁶ protoplasts/g of tissue and transfected with proteins and nucleotides, achieving delivery efficiencies up to 13.5%. The delivery of functional RNPs targeting phenylalanine ammonia lyase in P. taeda and phytoene desaturase in A. fraseri yielded gene editing efficiencies that reached 2.1% and 0.3%, respectively. This demonstration of RNP delivery for DNA-free genome editing in the protoplasts of P. taeda and A. fraseri illustrates the potential of CRISPR-Cas to enhance the traits of value in ecologically and economically important tree species. The editing system provides a foundation for future efforts to regenerate genome-edited forest trees to improve ecosystem health and natural resource sustainability. Full article

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is a promising biotechnology tool for genome editing. However, in living organisms, several pharmacokinetic challenges arise, including off-target side effects due to incorrect distribution, low bioavailability caused by membrane impermeability, and instability resulting from enzymatic degradation. Therefore, innovative delivery strategies must be developed to address these issues. Modified nanoparticles offer a potential solution for the non-invasive delivery of CRISPR/Cas9 ribonucleoproteins (Cas9 RNPs). Cas9 RNPs encapsulated in nanoparticles are protected from enzymatic degradation, similar to how microRNAs are shielded within exosomes. It is well-established that certain materials, including proteins, are expressed selectively in specific cell types. For example, the α-7 nicotinic receptor is expressed in endothelial and neuronal cells, while the αvβ3 integrin is expressed in cancer cells. These endogenous materials can facilitate receptor-mediated endocytosis or transcytosis. Nanoparticles encapsulating Cas9 RNPs and coated with ligands targeting such receptors may be internalized through receptor-mediated mechanisms. Once internalized, Cas9 RNPs could perform the desired gene editing in the nucleus after escaping the endosome through mechanisms such as the proton sponge effect or membrane fusion. In this review, I discuss the potential and advantages of delivering Cas9 RNP-encapsulated nanoparticles coated with ligands through receptor-mediated endocytosis or transcytosis. Full article

Lassa fever (LF), a viral hemorrhagic fever disease with a case fatality rate that can be over 20% among hospitalized LF patients, is endemic to many West African countries. Currently, no vaccines or therapies are specifically licensed to prevent or treat LF, hence the significance of developing therapeutics against the mammarenavirus Lassa virus (LASV), the causative agent of LF. We used in silico docking approaches to investigate the binding affinities of 2015 existing drugs to LASV proteins known to play critical roles in the formation and activity of the virus ribonucleoprotein complex (vRNP) responsible for directing replication and transcription of the viral genome. Validation of docking protocols were achieved with reference inhibitors of the respective targets. Our in silico docking screen identified five drugs (dexamethasone, tadalafil, mefloquine, ergocalciferol, and flunarizine) with strong predicted binding affinity to LASV proteins involved in the formation of the vRNP. We used cell-based functional assays to evaluate the antiviral activity of the five selected drugs. We found that flunarizine, a calcium-entry blocker, inhibited the vRNP activity of LASV and LCMV and virus surface glycoprotein fusion activity required for mammarenavirus cell entry. Consistently with these findings, flunarizine significantly reduced peak titers of LCMV in a multi-step growth kinetics assay in human A549 cells. Flunarizine is being used in several countries worldwide to treat vertigo and migraine, supporting the interest in exploring its repurposing as a candidate drug to treat LASV infections. Full article