Search Results (57)

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, a genome-editing technology pioneered in 2012, enables the precise correction of deleterious mutations or disruption of disease-causing genes through targeted double-strand breaks (DSBs), offering potential for treating genetic diseases. However, CRISPR/Cas9 can cause off-target cleavage at non-specific DNA sites, leading to unintended insertions or deletions (indels), which limit its safety and applicability despite ongoing improvements in specificity. Recently, prime editing (PE), an advanced CRISPR-derived technology, has been employed with a Cas9 nickase (Cas9n) fused with a reverse transcriptase and a prime editing guide RNA (pegRNA) to enable precise insertions, deletions, and transversions without inducing DSBs, thus reducing risks of indels and chromosomal aberrations. Furthermore, ongoing optimizations, such as improved pegRNA design and enhanced editing efficiency, have expanded the applications of PE in medical therapeutics, agriculture, and fundamental research. This review summarizes recent advancements in the PE system, including optimized pegRNA designs and enzyme engineering for enhanced efficiency and specificity, alongside novel delivery methods. It also evaluates cutting-edge delivery strategies, such as adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs) and novel extracellular vesicle (EV)-based systems, and explores PE applications in vitro and in vivo, including disease modeling and therapeutic gene correction. Full article

Microfluidic oxygenators promise to advance extracorporeal membrane oxygenation (ECMO) devices with enhanced hemodynamics and low prime volume. We are developing a silicon-based membrane oxygenator that will offer improved gas transfer and fluid flow control. Polyethylene glycol (PEG) has been used to improve hemocompatibility by providing excellent resistance to protein adsorption. Here, we characterized a polyethylene glycol surface modification of composite silicon–PDMS membranes to evaluate their effects on microfluidic oxygenator properties. X-ray photoelectron spectroscopy (XPS) and water contact angle goniometry confirmed successful PEG attachment, evidenced by the presence of characteristic C-O bonds and increased hydrophilicity, which was stable for 2 weeks. Oxygen flux tests demonstrated gas transfer rates as high as 89.6 ± 17.9 mL/min/m² and 50.8 ± 11.7 mL/min/m² for unmodified and PEG-coated membranes, respectively. Protein adsorption studies with human serum albumin (HSA) demonstrated a significant reduction in nonspecific protein binding on PEG-coated membranes with values as low as 14 ± 6 μg/cm². These studies expand on the characterization of our engineered oxygenator membranes and provide insight for the development of future surface optimization strategies to enhance hemocompatibility. Full article

Drought is a major abiotic stress that compromises seed germination, seedling establishment, and subsequent crop productivity, thereby threatening agricultural sustainability and food security. Developing effective seed-based strategies is therefore essential to enhance drought resilience. In this study, we investigated the efficacy of green-synthesized silver nanoparticles (AgNPs), produced using Azadirachta indica (neem) flower extract as a seed priming agent, to improve drought tolerance and early growth in wheat (Triticum aestivum). Seeds were primed with AgNPs (25–100 mg L⁻¹), PEG 6000 (−0.6, −0.8, and −1.0 MPa), and their combination (AgNPs + PEG 6000). AgNP priming enhanced germination by 72%, 86%, and 100% at 25, 50, and 75 mg L⁻¹, respectively, compared with the control, with 75 mg L⁻¹ identified as the optimal concentration. This treatment increased total chlorophyll and carotenoid contents by 14% and 6%, and elevated phenolic and flavonoid accumulation by 58% and 97%, respectively. Antioxidant enzyme activities were also substantially increased—catalase (CAT) by 44%, superoxide dismutase (SOD) by 23%, peroxidase (POX) by 11%, and glutathione reductase (GR) by 58%. Under drought stress, AgNP priming at 75 mg L⁻¹ improved germination by up to 80%, indicating enhanced drought tolerance. Elevated protein and antioxidant enzyme levels, along with reduced malondialdehyde (MDA), proline, and total soluble sugar levels, further confirmed mitigation of oxidative stress. Collectively, these results demonstrate that neem-mediated green-synthesized AgNPs could serve as an effective seed-priming agent, promoting wheat seedling establishment and enhancing drought resilience under water-deficit conditions. Full article

Ultrafine bubble (UFB) priming has recently emerged as a promising technique to enhance seed germination, yet its physiological mechanisms remain unclear. This study investigated the effects of UFB priming on adzuki bean (Vigna angularis) seeds under stress-free and drought conditions. Four treatments were tested: Control (non-primed), DW (primed with distilled water), UFB1 (4.56 × 10⁷ particles/mL), and UFB2 (1.13 × 10⁸ particles/mL). For priming, seeds were immersed in each solution at 25 ± 1 °C for 24 h. Seed traits, including water uptake, coat thickness, and total reactive oxygen species (ROS) levels in the priming solutions and seeds, were analyzed. UFB priming reduced seed coat thickness and slowed early water uptake without affecting final moisture content. Total ROS levels displayed a concentration-dependent pattern, with higher UFB increasing ROS in water but reducing them on the seed surface. Under stress-free conditions, all priming treatments accelerated germination, although higher UFB slightly prolonged T50. Under drought simulated by polyethylene glycol (PEG 10 and 15%), non-primed seeds were strongly inhibited, whereas primed seeds maintained high germination rates (>97%). Our results demonstrated that UFB priming improved water uptake, seed coat structure, and ROS signaling, enhancing seed performance of adzuki bean under favorable and drought conditions. Full article

Seed priming has become a promising technique in agriculture and crop-stress management. Several authors have shown that the positive effects of seed priming are associated with various metabolic, physiological, and biochemical modifications (enzyme activation, membrane repair, initiation of DNA/RNA, and protein synthesis) that enhance the speed, uniformity, and vigor of germination. However, the mechanisms underlying seed priming are not yet well understood. The aim of our work was to study the quantitative and qualitative metabolic changes in the embryonic axes (radicle and plumule) and cotyledons of Vigna unguiculata (L.) Walp. Seeds were subjected to osmopriming with polyethylene glycol (PEG), simple hydropriming, and double hydropriming (a novel treatment). Results indicated that all types of priming, particularly double hydropriming, strongly stimulated the hydrolysis of protein and carbohydrate reserves. This resulted in a decrease in soluble proteins and starch contents and an increase in amino acids and soluble sugars contents. Moreover, the priming promoted the biosynthesis of osmolytes such as proline and induced qualitative changes in the composition of amino acids and soluble sugars. These biochemical changes depend on the organ and treatment method applied to the seeds. It is worth noting that double hydropriming induces metabolic modifications to a greater extent than single hydropriming. Full article

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

In the case of direct sowing of rice in Japan, cold stress is a critical constraint affecting seed germination and early seedling development, ultimately reducing crop productivity. We evaluated the effects of priming, with or without iron coating on the germination and vigor of rice seeds harvested in 2022, 2023, and 2024. The assessments were conducted at seven temperature conditions: 13 °C, 15 °C, 17 °C, 19 °C, 21 °C, 23 °C, and 25 °C. Seeds were primed with or without PEG6000; coated with or without a mixture of calcined gypsum and iron powder; and tested for germination percentage, germination speed, and seedling vigor index. Under optimal conditions, iron-coated seeds harvested in 2022 showed a significant increase in germination from 58% (non-coated without priming) to 76% (coated with priming), and the seedling vigor index improved from 615 to 890. Under cold stress (15 °C), the coated seeds of the same year achieved 68% germination with priming compared to 46% in non-coated seeds without priming, with a vigor index increase from 480 to 750. Similar improvements were observed in seeds from 2023 and 2024, although the effect was more prominent in older than younger seeds. These results indicate that iron seed coating in combination with PEG priming mitigates the negative impacts of seed aging and enhances tolerance to cold stress during germination. The technique offers a promising, low-cost approach to improving rice establishment in environments facing suboptimal seed storage and early-season cold temperatures, in particular, aiming for direct sowing methods. Full article

Salt stress is a major constraint to crop productivity, negatively affecting plant physiology and fruit quality. This study hypothesized that seed priming with polyethylene glycol (PEG6000) might enhance antioxidant activity by mitigating oxidative stress in Solanum lycopersicum ‘Micro-Tom’ under salt stress. Seeds primed with –1.2 MPa PEG6000 were grown in Rockwool and treated with 0, 50, 100, 150, and 200 mM NaCl. Primed plants showed a 32% increase in leaf potassium (K⁺) and a 28% decrease in sodium (Na⁺) accumulation compared to non-primed plants under 150 mM NaCl. Glucose, fructose, and sucrose contents increased by 25%, 22%, and 19%, respectively, in primed fruits, while citric acid decreased by 15%. Malondialdehyde (MDA) and electrolyte leakage were reduced by 35% and 29%, respectively, in primed plants under moderate salinity. Antioxidant enzyme activities—SOD, POD, CAT, and APX were enhanced by 30–45% in primed plants under 100 and 150 mM NaCl, compared to non-primed controls. Abscisic acid (ABA) levels increased by 40% in primed roots under salt stress. Activities of polyamine-related enzymes (DAO, PAO, and ADC) also rose significantly. Priming improved protein content by 20% and relative water content by 18%. These results suggest that PEG6000 seed priming enhances salt tolerance by boosting antioxidant defense, regulating osmotic balance, and improving ion homeostasis, offering a viable strategy for sustaining tomato productivity under salinity. Full article

Cowpea is a nutritionally and economically valuable legume, known for its adaptability to adverse conditions. However, water stress negatively affects its development, requiring technologies to enhance resilience. This study aimed to induce tolerance to water deficit in cowpea through seed priming with polyethylene glycol 6000 (PEG 6000) and silicic acid. A completely randomized experiment was conducted in a phytotron chamber with two water regimes (W50 and W100) and six seed priming treatments, with four replications. Priming consisted of three water potentials induced by PEG 6000 (0 MPa, −0.4 MPa, and −0.8 MPa) and two silicon concentrations (0 and 200 mg L⁻¹). Gas exchange parameters, including photosynthetic rate (A), transpiration rate (E), stomatal conductance (gs), intercellular CO₂ concentration (Ci), instantaneous water use efficiency (WUEi), and instantaneous carboxylation efficiency (iCE), were evaluated. Seed priming with PEG 6000 and silicon improved A, WUEi, and iCE under water deficit. Treatments 2 (0 MPa + 200 mg L⁻¹ Si), 3 (−0.4 MPa + 0 mg L⁻¹ Si), and 4 (−0.4 MPa + 200 mg L⁻¹ Si) enhanced gas exchange, suggesting an effective strategy for improving drought tolerance in cowpea and ensuring food security. Full article

Drought stress is one of the key environmental factors restricting the germination of alfalfa seeds (Medicago sativa L.). Nanopriming is an innovative seed-priming technology able to meet economic, agronomic, and environmental needs in agriculture. However, the use of conventional nanomaterials is hampered by high costs, environmental risks, and biotoxicity. In this study, we synthesized iron oxide nanoparticles (Fe₃O₄NPs) using seasonal Ginkgo biloba leaf extracts (collected from August to November) obtained via an enzymatic ultrasonic-assisted method. The synthesized Fe₃O₄NPs were characterized using SEM, EDS, DLS, FTIR, UV-Vis, and XRD. To investigate the effects of Fe₃O₄NP priming on alfalfa seed germination under drought stress, germination and pot experiments were conducted with five Fe₃O₄NP priming concentrations (unprimed, 0, 20, 40, and 60 mg/L) and three PEG-6000 concentrations (0%, 10%, and 15%) to simulate normal, moderate, and severe drought conditions. The results showed that leaf extracts collected in November exhibited the highest flavonoid content (12.8 mg/g), successfully yielding bioactive-capped spherical Fe₃O₄NPs with a particle size of 369.5 ± 100.6 nm. Germination experiments revealed that under severe drought stress (15% PEG-6000), the 40 mg/L Fe₃O₄NP treatment most effectively enhanced seed vigor, increasing the germination rate, vigor index, and α-amylase activity by 22.1%, 189.4%, and 35.5% (p < 0.05), respectively, compared to controls. Under moderate drought stress (10% PEG-6000), the 20 mg/L Fe₃O₄NP treatment optimally improved germination traits, increasing the germination rate by 25.5% and seedling elongation by 115.6%. The pot experiments demonstrated morphological adaptations in alfalfa seedlings: under moderate drought stress, the 40 mg/L Fe₃O₄NPs significantly increased lateral root numbers, while under severe drought stress, the 60 mg/L Fe₃O₄NPs increased the root surface area by 20.5% and preserved the roots’ structural integrity compared to controls. These findings highlight that Fe₃O₄NPs synthesized via Ginkgo leaf extracts and enzymatic ultrasonic methods exhibit promising agricultural potential. The optimal Fe₃O₄NP priming concentrations enhanced seed vigor, germination traits, and drought resistance by modulating root morphology, with concentration-specific efficacy under varying drought intensities. Full article

Prime editing (PE) is a CRISPR-based tool for genome engineering that can be applied to generate human induced pluripotent stem cell (hiPSC)-based disease models. PE technology safely introduces point mutations, small insertions, and deletions (indels) into the genome. It uses a Cas9-nickase (nCas9) fused to a reverse transcriptase (RT) as an editor and a PE guide RNA (pegRNA), which introduces the desired edit with great precision without creating double-strand breaks (DSBs). PE leads to minimal off-targets or indels when introducing single-strand breaks (SSB) in the DNA. Low efficiency can be an obstacle to its use in hiPSCs, especially when the genetic context precludes the screening of multiple pegRNAs, and other strategies must be employed to achieve the desired edit. We developed a PE platform to efficiently generate isogenic models of Mendelian disorders. We introduced the c.25G>A (p.V9M) mutation in the NMNAT1 gene with over 25% efficiency by optimizing the PE workflow. Using our optimized system, we generated other isogenic models of inherited retinal diseases (IRDs), including the c.1481C>T (p.T494M) mutation in PRPF3 and the c.6926A>C (p.H2309P) mutation in PRPF8. We modified several determinants of the hiPSC PE procedure, such as plasmid concentrations, PE component ratios, and delivery method settings, showing that our improved workflow increased the hiPSC editing efficiency. Full article

Prime editor, an editing tool based on the CRISPR/Cas9 system, allows for all 12 types of nucleotide exchanges and arbitrary indels in genomic sequences without the need for inducing DNA double-strand breaks. Despite its flexibility and precision, prime editing efficiency is still low and hindered by various factors such as target sites, editing types, and the length of the primer binding site. In this study, we developed a prime editing system by incorporating an RNA motif at the 3′ terminal of the pegRNA and integrating all twin prime editor factors into a single plasmid. These two strategies enhanced prime editing efficiency at target sites by up to 3.58-fold and 2.19-fold, respectively. Subsequently, enhanced prime editor was employed in goat cells and embryos to efficiently insert a 38 bp attB sequence into the Gt(ROSA)26Sor (Rosa26) and C-C motif chemokine receptor 5 (CCR5) loci. The enhanced prime editor can mediate 11.9% and 6.8% editing efficiency in parthenogenetic activation of embryos through embryo microinjection. In summary, our study introduces a modified prime editing system with improved editing and transfection efficiency, making it more suitable for inserting foreign sequences into primary cells and embryos. These results broaden the potential applications of prime editing technologies in the production of transgenic animals. Full article

Seed germination (GS) and seedling growth are vital plant stages that can be affected by stresses such as drought and aging, which cause deterioration and reduce seed viability. With the aim of homogenizing and improving GS, priming treatments with biostimulants such as the antioxidant melatonin are commonly used in seeds. In this study, the effects of melatonin on germination and seedling growth in two different situations, i.e., aging seeds of rice, barley, and sorghum and under polyethylene glycol (PEG)-induced drought stress in sorghum, were studied. Aged seeds were primed for 7 days in different concentrations of melatonin, and drought stress seeds were primed for 24 h before PEG treatment for 7 days, and germination and initial growth parameters were monitored. Aging-seeds of rice and barley showed the maximum response in terms of germination percentage at 20 µM melatonin and 0.05 µM respectively; while aging-seeds of sorghum showed improvement in germination for practically all concentrations studied, even the highest tested at 50 µM. Regarding the effect of melatonin treatments on drought stress in sorghum seeds, all the studied parameters showed a significant attenuation of the adverse effects of drought stress, alleviating them, for all concentrations tested but especially at 200 µM melatonin. The results obtained confirm that priming seeds with melatonin under low germinability conditions relieves stress and improves both germination and seedling growth. Full article

Prime editing shows potential as a precision genome editing technology, as well as the potential to advance the development of next-generation nanomedicine for addressing neurological disorders. However, turning in prime editors (PEs), which are macromolecular complexes composed of CRISPR/Cas9 nickase fused with a reverse transcriptase and a prime editing guide RNA (pegRNA), to the brain remains a considerable challenge due to physiological obstacles, including the blood–brain barrier (BBB). This review article offers an up-to-date overview and perspective on the latest technologies and strategies for the precision delivery of PEs to the brain and passage through blood barriers. Furthermore, it delves into the scientific significance and possible therapeutic applications of prime editing in conditions related to neurological diseases. It is targeted at clinicians and clinical researchers working on advancing precision nanomedicine for neuropathologies. Full article

One of the main climate change-related variables limiting agricultural productivity that ultimately leads to food insecurity appears to be drought. With the use of a recently discovered nanopriming technology, seeds can endure various abiotic challenges. To improve seed quality and initial growth of 8-day-old field pea seedlings (cv. NS Junior) under optimal and artificial drought (PEG-induced) laboratory conditions, this study aimed to assess the efficacy of priming with three different nanomaterials: Nanoplant Ultra (Co, Mn, Cu, Fe, Zn, Mo, and Se), Nanoplant Ca-Si (Ca, Si, B, and Fe), and Nanoplant Sulfur (S). The findings indicate that nanopriming seed treatments have a positive impact on seed quality indicators, early plant growth, and drought resilience in field pea plants established in both optimal and drought-stressed conditions. Nevertheless, all treatments showed a positive effect, but their modes of action varied. Nanoplant Ultra proved to be the most effective under optimal conditions, whereas Nanoplant Ca-Si and Nanoplant Sulfur were the most efficient under drought stress. After a field evaluation, the examined comprehensive nanomaterials may be utilized as priming agents for pea seed priming to boost seed germination, initial plant growth, and crop productivity under various environmental conditions. Full article