DNA Delivery Research Articles

The design and engineering of synthetic genomes.

Synthetic genomics seeks to design and construct entire genomes to mechanistically dissect fundamental questions of genome function and to engineer organisms for diverse applications, including bioproduction of high-value chemicals and biologics, advanced cell therapies, and stress-tolerant crops. Recent progress has been fuelled by advancements in DNA synthesis, assembly, delivery and editing. Computational innovations, such as the use of artificial intelligence to provide prediction of function, also provide increasing capabilities to guide synthetic genome design and construction. However, translating synthetic genome-scale projects from idea to implementation remains highly complex. Here, we aim to streamline this implementation process by comprehensively reviewing the strategies for design, construction, delivery, debugging and tailoring of synthetic genomes as well as their potential applications.

Electroporation-mediated novel albumin-fused Flt3L DNA delivery promotes cDC1-associated anticancer immunity.

Dendritic cells (DCs) constitute a distinct type of immune cell found within tumors, serving a central role in mediating tumor antigen-specific immunity against cancer cells. Frequently, DC functions are dysregulated by the immunosuppressive signals present within the tumor microenvironment (TME). Consequently, DC manipulation holds great potential to enhance the cytotoxic T cell response against cancer diseases. One strategy involves administering Fms-like tyrosine kinase receptor 3 ligand (Flt3L), a vitally important cytokine for DC development. In this current study, the electroporation-mediated delivery of a novel albumin-fused Flt3L DNA (alb-Flt3L DNA) demonstrated the ability to induce an anti-tumor immune response. This albumin fusion construct possesses more persistent bioactivity in targeted organs. Furthermore, TC-1-bearing-C57BL/6 mice receiving alb-Flt3L DNA treatment presented better tumor control and superior survival. Cellular analysis revealed that alb-Flt3L DNA administration promoted robust DC and cDC1 expansion. In addition, increased levels of IFN-γ-secreting CD8+ lymphocytes were found in correlation to greater cDC1 population. Moreover, the toxicity of alb-Flt3L administration is limited. Collectively, our data showcases a novel DC-based immunotherapy using electroporation to administer alb-Flt3L DNA.

Efficient Polymeric Nanoparticle Gene Delivery Enabled Via Tri- and Tetrafunctional Branching.

Poly(β-amino ester) (PBAE) nanoparticles (NPs) show great promise for nonviral gene delivery. Recent studies suggest branched PBAEs (BPBAEs) offer advantages over linear counterparts, but the effect of polymer structure has not been well investigated across many chemical constituents. Here, a library of BPBAEs was synthesized with tri- and tetrafunctional branching. These polymers self-assemble with DNA to form highly cationic, monodisperse NPs with notably small size (∼50 nm). Optimal transfection occurred with polymer structures that featured moderate PBAE branching, enabling complete DNA encapsulation, rapid NP uptake, and robust expression at low DNA doses and polymer amounts. Optimized NPs enabled efficient DNA delivery to diverse cell types in vitro while maintaining high cellular viability, demonstrating significant improvements over a well-performing linear PBAE counterpart. BPBAEs also facilitated efficient mRNA and siRNA delivery, highlighting the versatility of these structures and demonstrating the broad utility of BPBAE NPs as vectors for nucleic acid delivery.

Efficient electroporation in primary cells with PEDOT:PSS electrodes.

Precise and efficient delivery of macromolecules into cells enhances basic biology research and therapeutic applications in cell therapies, drug delivery, and personalized medicine. While pulsed electric field electroporation effectively permeabilizes cell membranes to deliver payloads without the need for toxic chemical or viral transduction agents, conventional bulk electroporation devices face major challenges with cell viability and heterogeneity due to variations in fields generated across cells and electrochemistry at the electrode-electrolyte interface. Here, we introduce the use of microfabricated electrodes based on the conducting polymer poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS), which substantially increases cell viability and transfection efficiency. As a proof of concept, we demonstrate the enhanced delivery of Cas9 protein, guide RNA, and plasmid DNA into cell lines and primary cells. This use of PEDOT:PSS enables rapid modification of difficult-to-transfect cell types to accelerate their study and use as therapeutic platforms.

Structuring lipid nanoparticles, DNA, and protein corona into stealth bionanoarchitectures for in vivo gene delivery

Lipid nanoparticles (LNPs) play a crucial role in addressing genetic disorders, and cancer, and combating pandemics such as COVID-19 and its variants. Yet, the ability of LNPs to effectively encapsulate large-size DNA molecules remains elusive. This is a significant limitation, as the successful delivery of large-size DNA holds immense potential for gene therapy. To address this gap, the present study focuses on the design of PEGylated LNPs, incorporating large-sized DNA, departing from traditional RNA and ionizable lipids. The resultant LNPs demonstrate a unique particle morphology. These particles were further engineered with a DNA coating and plasma proteins. This multicomponent bionanoconstruct exhibits enhanced transfection efficiency and safety in controlled laboratory settings and improved immune system evasion in in vivo tests. These findings provide valuable insights for the design and development of bionanoarchitectures for large-size DNA delivery, opening new avenues for transformative gene therapies.

Iontophoresis and electroporation-assisted microneedles: advancements and therapeutic potentials in transdermal drug delivery.

Transdermal drug delivery (TDD) using electrically assisted microneedle (MN) systems has emerged as a promising alternative to traditional drug administration routes. This review explores recent advancements in this technology across various therapeutic applications. Integrating iontophoresis (IP) and electroporation (EP) with MN technology has shown significant potential in improving treatment outcomes for various conditions. Studies demonstrate their effectiveness in enhancing vaccine and DNA delivery, improving diabetes management, and increasing efficacy in dermatological applications. The technology has also exhibited promise in delivering nonsteroidal anti-inflammatory drugs (NSAIDs), treating multiple sclerosis, and advancing obesity and cancer therapy. These systems offer improved drug permeation, targeted delivery, and enhanced therapeutic effects. While challenges remain, including safety concerns and technological limitations, ongoing research focuses on optimizing these systems for broader clinical applications. The future of electrically assisted MN technologies in TDD appears promising, with potential advancements in personalized medicine, smart monitoring systems, and expanded therapeutic applications.

Optimizing Transfection Efficiency of Spermine Polar Head Cholesterol-Based Cationic Lipids with Amino Acid Linker.

In this work, a series of spermine polar head cholesterol-based cationic lipids with various amino acid spacers were synthesized and evaluated as non-viral gene delivery systems. The physicochemical properties of the resulting lipoplexes, formed from these lipids and DOPE, were assessed, including zeta-potential, DNA binding and DNA protection from serum. Transfection efficiency and cytotoxicity were examined under serum-free and 10-40 % serum-containing conditions. The results showed that the physicochemical properties of cationic lipids, both with and without amino acid spacers, were not significantly different. Cationic liposomes composed of lipid Sper-Ahx-Chol, which has a 6-aminohexanoic acid spacer, and DOPE exhibited greater transfection efficiency in HeLa cells compared to Lipofectamine3000, both in the absence and presence of 10-40 % serum. Additionally, lipid Sper-His-Chol with a histidine spacer and Sper-Ahx-Chol showed higher efficiency than Lipofectamine3000 against HEK293T under 40 % serum conditions. These results suggest that the incorporation of amino acids into the cationic lipids can significantly enhance their DNA delivery efficiency. Specifically, certain amino acid modifications improved transfection efficiency while maintaining low cytotoxicity. Our findings highlight the potential of amino acid-tailored cationic lipids as promising vectors for enhanced DNA delivery.

Remote-Controlled Gene Delivery in Coaxial 3D-Bioprinted Constructs using Ultrasound-Responsive Bioinks

IntroductionCoaxial 3D bioprinting has advanced the formation of tissue constructs that recapitulate key architectures and biophysical parameters for in-vitro disease modeling and tissue-engineered therapies. Controlling gene expression within these structures is critical for modulating cell signaling and probing cell behavior. However, current transfection strategies are limited in spatiotemporal control because dense 3D scaffolds hinder diffusion of traditional vectors. To address this, we developed a coaxial extrusion 3D bioprinting technique using ultrasound-responsive gene delivery bioinks. These bioink materials incorporate echogenic microbubble gene delivery particles that upon ultrasound exposure can sonoporate cells within the construct, facilitating controllable transfection.MethodsPhospholipid-coated gas-core microbubbles were electrostatically coupled to reporter transgene plasmid payloads and incorporated into cell-laden alginate bioinks at varying particle concentrations. These bioinks were loaded into the coaxial nozzle core for extrusion bioprinting with CaCl2 crosslinker in the outer sheath. Resulting bioprints were exposed to 2.25 MHz focused ultrasound and evaluated for microbubble activation and subsequent DNA delivery and transgene expression.ResultsCoaxial printing parameters were established that preserved the stability of ultrasound-responsive gene delivery particles for at least 48 h in bioprinted alginate filaments while maintaining high cell viability. Successful sonoporation of embedded cells resulted in DNA delivery and robust ultrasound-controlled transgene expression. The number of transfected cells was modulated by varying the number of focused ultrasound pulses applied. The size region over which DNA was delivered was modulated by varying the concentration of microbubbles in the printed filaments.ConclusionsOur results present a successful coaxial 3D bioprinting technique designed to facilitate ultrasound-controlled gene delivery. This platform enables remote, spatiotemporally-defined genetic manipulation in coaxially bioprinted tissue constructs with important applications for disease modeling and regenerative medicine.

Correction: Geng et al. Intracellular Delivery of DNA and Protein by a Novel Cell-Permeable Peptide Derived from DOT1L. Biomolecules 2020, 10, 217

In the original article [...]

Plasmid DNA Delivery into the Skin via Electroporation with a Depot-Type Electrode.

Objectives: Non-viral mediated plasmid DNA transfection by electroporation (EP) is an established method for gene transfection. In this study, the usefulness of direct EP at an intradermal (i.d.) site (DEP) with implanted electrodes to achieve a high protein expression level was investigated. In addition, DEP application with various intervals with a low application voltage was also evaluated to confirm its effect on protein expression. Methods: Green fluorescent protein (GFP)- and luciferase-encoding DNA were administrated, and GFP and luciferase were evaluated. Results: A higher protein expression level was observed after green fluorescent protein (GFP)- and luciferase-encoding DNA were delivered by i.d. injection followed by DEP application. When luciferase expression was observed with an in vivo imaging system, continuous expression was confirmed over 21 days after i.d. injection followed by DEP at 100 V. This approach provided increased gene expression levels compared with conventional EP methods via the stratum corneum layer. In addition, the effect of application voltage on luciferase expression was investigated; two-time applications (repeated DEP) at 20 V with 5 min intervals showed similar luciferase expression level to single DEP application with 100 V. Histological observations showed the skin became thicker after a single DEP at 100 V, whereas no apparent thickness changes were confirmed after repeated DEP at 20 V with 5 min intervals. Conclusions: This study revealed that direct i.d. voltage application achieved high protein expression levels even at low voltages. Skin is a promising administration site for DNA vaccines, so this approach may be effective for DNA vaccine delivery into skin tissue.

Comparison of ionizable lipids for lipid nanoparticle mediated DNA delivery

Lipid nanoparticles (LNPs) are successfully used for RNA-based gene delivery. In the context of gene replacement therapies, however, delivery of DNA expression plasmids using LNPs as a non-viral vector could be a promising strategy for the induction of longer-lasting effects.Therefore, DNA expression plasmids (3 to 4 kbp) coding for fluorescent markers or luciferase were combined with LNPs. Different clinically used ionizable lipids (DLin-MC3-DMA, SM-102, and ALC-0315) were tested to compare their influence on DNA plasmid delivery.DNA-LNPs were characterized with respect to their colloidal properties (size, polydispersity, ζ-potential, morphology), in vitro performance (cellular uptake, DNA delivery, and gene expression), and in vivo characteristics (biodistribution and luciferase gene expression). At an optimized N/P ratio of 6, spherical, small and monodisperse particles with anionic ζ-potential were obtained. Efficient transgene expression was achieved with a minimum amount of 1 pg DNA per initially plated cells. Zebrafish studies allowed selection of DNA-LNPs, which demonstrated prolonged blood circulation, avoidance of macrophage clearance, and vascular extravasation.Our comparative study demonstrates a high impact of the ionizable lipid type on DNA-LNP performance. Superior transfection efficiency of DNA-LNPs containing the ionizable lipid ALC-0315 was confirmed in wildtype mice.

Utilization of chitosan nanocomposites loaded with quantum dots enables efficient and traceable DNA delivery

Chitosan is widely employed in gene carriers due to its excellent gene loading capacity, ease of modification, and exceptional biodegradability. However, low gene delivery efficiency, high cytotoxicity, and lack of tracer biomimetic properties limit its clinical use. To address these issues, a novel biomimetic tracking gene delivery carrier, RBCm-C50kQT, was constructed by using the design scheme of cell membrane coated carbon quantum dots/chitosan. This carrier improves stability and tracking performance while embedding the cell membrane enhances biosafety. RBCm-C50kQT effectively carries and protects DNA, improving uptake and transfection efficiency with reduced cytotoxicity. It maintains strong fluorescence tracking and shows high uptake efficiencies of 83.62 % and 77.45 % in 293 T and HeLa cells, respectively, with maximum transfection efficiencies of 68.80 % and 45.47 %. This advancement supports gene therapy improvements and paves the way for future clinical applications.

Investigation of Mother-to-Child Transmission of Hepatitis B in Yinchuan, China: Cross-Sectional Survey Study.

Hepatitis B poses a significant global public health challenge, with mother-to-child transmission (MTCT) being the primary method of hepatitis B virus (HBV) transmission. The prevalence of HBV infection in China is the highest in Asia, and it carries the greatest burden globally. This study aims to critically evaluate the existing local strategies for preventing MTCT and the proposed potential enhancements by analyzing the prevalence of hepatitis B among pregnant women and their neonates in Yinchuan. From January 2017 to December 2021, 37,557 prenatal screening records were collected. Among them, 947 pregnant women who tested positive for hepatitis B surface antigen (HBsAg) near delivery and their 960 neonates were included in an HBV-exposed group, while 29 pregnant women who tested negative and their 30 neonates were included in an HBV-nonexposed group. HBV markers in maternal peripheral blood and neonatal cord blood were analyzed using the least absolute shrinkage and selection operator (LASSO) regression, logistic regression, chi-square test, t-test, and U-test. Additionally, to further evaluate the diagnostic value of HBsAg positivity in cord blood, we conducted an additional follow-up study on 103 infants who tested positive for HBsAg in their cord blood. The prevalence of HBV among pregnant women was 2.5% (947/37,557), with a declining trend every year (χ²4=19.7; P=.001). From 2018 to 2020, only 33.0% (35/106) of eligible pregnant women received antiviral medication treatment. Using LASSO regression to screen risk factors correlated with HBsAg positivity in cord blood (when log [λ] reached a minimum value of -5.02), 5 variables with nonzero coefficients were selected, including maternal hepatitis B e-antigen (HBeAg) status, maternal hepatitis B core antibody (HBcAb) status, maternal HBV DNA load, delivery method, and neonatal birth weight. Through univariate and multivariate logistic regression, delivery by cesarean section (adjusted odds ratio [aOR] 0.52, 95% CI 0.31-0.87), maternal HBeAg positivity (aOR 2.05, 95% CI 1.27-3.33), low maternal viral load (aOR 2.69, 95% CI 1.33-5.46), and high maternal viral load (aOR 2.69, 95% CI 1.32-5.51) were found to be strongly associated with cord blood HBsAg positivity. In the additional follow-up study, 61 infants successfully completed the follow-up, and only 2 were found to be infected with HBV. The mothers of both these infants had detectable HBV DNA levels and should have received standard antiviral therapy. The results of the hepatitis B surface antibody (HBsAb) positivity rate and titer test indicated a gradual decline in the immunity of vaccinated infants as the interval after vaccination increased. The clinical relevance of HBV marker detection in cord blood is restricted within the current prevention measures for MTCT. There is an emphasis on the significance of public education regarding hepatitis B and the reinforcement of postnatal follow-up for the prevention of MTCT.

Targeted Delivery of Circular Single-Stranded DNA Encoding IL-12 for the Treatment of Triple-Negative Breast Cancer.

Interleukin-12 (IL-12) is a critical cytokine with notable anticancer properties, including enhancing T-cell-mediated cancer cell killing, and curbing tumor angiogenesis. To date, many approaches are evaluated to achieve in situ overexpression of IL-12, minimizing leakage and the ensuing toxicity. Here, it is focused on circular single-stranded DNA (Css DNA), a type of DNA characterized by its unique structure, which could be expressed in mammals. It is discovered that Css DNA can induce sustained luciferase expression for half a year by intramuscular injection and showed effective antitumor results by intratumoral injection. Motivated by these findings, a folate-modified LNP system is now developed for the delivery of Css DNA expressing IL-12 for the therapy of 4T1 triple-negative breast cancer (TNBC). This delivery system effectively activates anti-cancer immune responses, slows tumor growth, significantly prolongs survival in animal models, and prevents tumor recurrence. After 6 months of long-term observation, the elevated level of IL-12 is still detectable in the lymph nodes and serum of the cured mice. This study highlights the long-term sustained expression capacity of Css DNA and its ability to inhibit recurrence, and the potential of tumor-targeted LNPs for Css DNA-based cancer therapy, providing a new insight into gene overexpression strategy.

Targeting Death Receptor 5 (DR5) for the imaging and treatment of primary bone and soft tissue tumors: an update of the literature.

Death Receptor 5 (DR5) is expressed on the surface of primary bone and soft tissue sarcoma cells, and its activation induces cell death primarily through apoptosis. The combination of DR5 agonists and commonly used chemotherapeutic agents, such as doxorubicin, can promote cell death. Currently, clinical trials are investigating the effectiveness of DR5 activation using new biological agents, such as bi-specific or tetravalent antibodies, in improving the survival of patients with relapsed or refractory cancers. Furthermore, investigations continue into the use of novel combination therapies to enhance DR5 response, for example, with inhibitor of apoptosis protein (IAP) antagonist agents [such as the second mitochondria-derived activator of caspase (SMAC) mimetics] and with immune checkpoint inhibitor anti-programmed death-ligand 1 (anti-PD-L1) or anti-programmed cell death-1 (anti-PD-1) antibodies. Other therapies include nanoparticle-mediated delivery of TRAIL plasmid DNA or TRAIL mRNA and stem cells as a vehicle for the targeted delivery of anti-cancer agents, such as TRAIL, to the tumor. Scoping review of the literature from November 2017 to March 2024, utilizing PubMed and Google Scholar. New agents under investigation include nanoTRAIL, anti-Kv10.1, multimeric IgM, and humanized tetravalent antibodies. Developments have been made to test novel agents, and imaging has been used to detect DR5 in preclinical models and patients. The models include 3D spheroids, genetically modified mouse models, a novel jaw osteosarcoma model, and patient-derived xenograft (PDX) animal models. There are currently two ongoing clinical trials focusing on the activation of DR5, namely, IGM-8444 and INBRX-109, which have progressed to phase 2. Further modifications of TRAIL delivery with fusion to single-chain variable fragments (scFv-TRAIL), directed against tumor-associated antigens (TAAs), and in the use of stem cells focus on targeted TRAIL delivery to cancer cells using bi-functional strategies. In vitro, in vivo, and clinical trials, as well as advances in imaging and theranostics, indicate that targeting DR5 remains a valid strategy in the treatment of some relapsed and refractory cancers.

Potential applications of PEI loaded graphene oxide quantum dots in safe and efficient DNA delivery

In order to improve the safety and efficacy of gene delivery using polyethyleneimine (PEI), this work focused on developing PEI quantum dots and incorporating them onto graphene to improve gene loading capacity. The successful creation of graphene-polyethyleneimine quantum dots (GPQDs) was confirmed through fluorescence emission spectrum and infrared spectroscopy. Both polyethyleneimine quantum dots (PQDs) and GPQDs exhibited blue fluorescence when excited between 310 and 430 nm, indicating that they retained the fluorescence characteristics of broad excitation and narrow emission. Furthermore, GPQDs displayed smaller particle sizes, increased hydrophilicity, and better dispersibility compared to PQDs. GPQDs were able to efficiently load DNA with lower cytotoxicity than PQDs and PEI. The enhanced fluorescence properties of GPQDs allowed for the observation that GPQDs/DNA complexes were effectively internalized by cells with higher uptake efficiency than PEI/DNA and PQDs/DNA complexes. The study demonstrated that GPQDs/DNA complexes successfully transfected 293T and HeLa cells, with GPQDs prepared using PEI25k showing improved cell uptake and transfection efficiency compared to those prepared with PEI10k. Overall, the findings suggest that GPQDs are a more effective and safer gene delivery vector than PEI, with promising potential for future gene delivery applications.

Safe and effective in vivo delivery of DNA and RNA using proteolipid vehicles

Genetic medicines show promise for treating various diseases, yet clinical success has been limited by tolerability, scalability, and immunogenicity issues of current delivery platforms. To overcome these, we developed a proteolipid vehicle (PLV) by combining features from viral and non-viral approaches. PLVs incorporate fusion-associated small transmembrane (FAST) proteins isolated from fusogenic orthoreoviruses into a well-tolerated lipid formulation, using scalable microfluidic mixing. Screening a FAST protein library, we identified a chimeric FAST protein with enhanced membrane fusion activity that improved gene expression from an optimized lipid formulation. Systemically administered FAST-PLVs showed broad biodistribution and effective mRNA and DNA delivery in mouse and non-human primate models. FAST-PLVs show low immunogenicity and maintain activity upon repeat dosing. Systemic administration of follistatin DNA gene therapy with FAST-PLVs raised circulating follistatin levels and significantly increased muscle mass and grip strength. These results demonstrate the promising potential of FAST-PLVs for redosable gene therapies and genetic medicines.

Degradation of polyethylene terephthalate (PET) plastics by wastewater bacteria engineered via conjugation.

Wastewater treatment plants are one of the major pathways for microplastics to enter the environment. In general, microplastics are contaminants of global concern that pose risks to ecosystems and human health. Here, we present a proof-of-concept for reduction of microplastic pollution emitted from wastewater treatment plants: delivery of recombinant DNA to bacteria in wastewater to enable degradation of polyethylene terephthalate (PET). Using a broad-host-range conjugative plasmid, we enabled various bacterial species from a municipal wastewater sample to express FAST-PETase, which was released into the extracellular environment. We found that FAST-PETase purified from some transconjugant isolates could degrade about 40% of a 0.25 mm thick commercial PET film within 4 days at 50°C. We then demonstrated partial degradation of a post-consumer PET product over 5-7 days by exposure to conditioned media from isolates. These results have broad implications for addressing the global plastic pollution problem by enabling environmental bacteria to degrade PET.

Lipid-Based Nanoparticles Fused with Natural Killer Cell Plasma Membrane Proteins for Triple-Negative Breast Cancer Therapy.

Immunotherapy combined with chemicals and genetic engineering tools is emerging as a promising strategy to treat triple-negative breast cancer (TNBC), which is more aggressive with poorer progress than other breast cancer subtypes. In this study, lipid-based nanoparticles (LNPs) possessed an NK cell-like function that could deliver tumor-specific therapeutics and inhibit tumor growth. LNPs fused with an NK cell membrane protein system (NK-LNP) have three main features: (i) hydrophilic plasmid DNA can inhibit TNBC metastasis when encapsulated within LNPs and delivered to cells; (ii) the lipid composition of LNPs, including C18 ceramide, exhibits anticancer effects; (iii) NK cell membrane proteins are immobilized on the LNP surface, enabling targeted delivery to TNBC cells. These particles facilitate the targeted delivery of HIC1 plasmid DNA and the modulation of immune cell functions. Delivered therapeutic genes can inhibit metastasis of TNBC and then induce apoptotic cell death while targeting macrophages to promote cytokine release. The anticancer effect is expected to be applied in treating various difficult-to-treat cancers with LNP fused with NK cell plasma membrane proteins, which can simultaneously deliver therapeutic chemicals and genes.

Fusiform nanoparticle boosts efficient genetic transformation in Sclerotinia sclerotiorum

BackgroundSclerotinia sclerotiorum is a highly destructive phytopathogenic fungus that poses a significant threat to a wide array of crops. The current constraints in genetic manipulation techniques impede a thorough comprehension of its pathogenic mechanisms and the development of effective control strategies.ResultsHerein, we present a highly efficient genetic transformation system for S. sclerotiorum, leveraging the use of fusiform nanoparticles, which are synthesized with FeCl3 and 2,6-diaminopyrimidine (DAP). These nanoparticles, with an average longitude length of 59.00 nm and a positively charged surface, facilitate the direct delivery of exogenous DNA into the mycelial cells of S. sclerotiorum, as well as successful integration with stable expression. Notably, this system circumvents fungal protoplast preparation and tedious recovery processes, streamlining the transformation process considerably. Furthermore, we successfully employed this system to generate S. sclerotiorum strains with silenced oxaloacetate acetylhydrolase-encoding gene Ss-oah1.ConclusionsOur findings demonstrate the feasibility of using nanoparticle-mediated delivery as a rapid and reliable tool for genetic modification in S. sclerotiorum. Given its simplicity and high efficiency, it has the potential to significantly propel genetic research in filamentous fungi, offering new avenues for elucidating the intricacies of pathogenicity and developing innovative disease management strategies.Graphical