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Climate change and the exploration of new areas of cultivation have impacted the yields of several economically important crops worldwide. Both conventional plant breeding based on planned crosses between parents with specific traits and genetic engineering to develop new biotechnological tools (NBTs) have allowed the development of elite cultivars with new features of agronomic interest. The use of these NBTs in the search for agricultural solutions has gained prominence in recent years due to their rapid generation of elite cultivars that meet the needs of crop producers, and the efficiency of these NBTs is closely related to the optimization or best use of their elements. Currently, several genetic engineering techniques are used in synthetic biotechnology to successfully improve desirable traits or remove undesirable traits in crops. However, the features, drawbacks, and advantages of each technique are still not well understood, and thus, these methods have not been fully exploited. Here, we provide a brief overview of the plant genetic engineering platforms that have been used for proof of concept and agronomic trait improvement, review the major elements and processes of synthetic biotechnology, and, finally, present the major NBTs used to improve agronomic traits in socioeconomically important crops.

Agrobacterium‐mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium‐mediated transformation has three major steps in laboratory‐controlled experiments: the delivery of T‐DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium‐mediated plant transformation. It has been reported that increasing the number of cells transformed by T‐DNA delivery can improve the frequency of stable transformation. Previously, we demonstrated that a reduction in ethylene production by plant cells during cocultivation with A. tumefaciens‐expressing 1‐aminocyclopropane‐1‐carboxylic acid (ACC) deaminase resulted in increased T‐DNA delivery into the plant cells. In this study, to further improve T‐DNA delivery by A. tumefaciens, we modified the expression cassette of the ACC deaminase gene using vir gene promoter sequences. The ACC deaminase gene driven by the virD1 promoter was expressed at a higher level, resulting in a higher ACC deaminase activity in this A. tumefaciens strain than in the strain with the lac promoter used in a previous study. The newly developed A. tumefaciens strain improves the delivery of T‐DNA into Solanum lycopersicum (tomato) and Erianthus ravennae plants and thus may be a powerful tool for the Agrobacterium‐mediated genetic engineering of plants. Agrobacterium‐mediated transformation is a useful tool for the genetic modification of plants, although its efficiency is low for several plant species. The newly developed Agrobacterium tumefaciens with improved ACC deaminase activity showed improved T‐DNA delivery into S. lycopersicum and E. ravennae and thus may be a powerful tool for Agrobacterium‐mediated genetic engineering of plants.

Conventional approaches like Agrobacterium -mediated transformation, viral transduction, biolistic particle bombardment, and polyethylene glycol (PEG)-facilitated delivery methods have been optimized for transporting specific genes to various plant cells. These conventional approaches in genetically modified crops are dependent on several factors like plant types, cell types, and genotype requirements, as well as numerous disadvantages such as time-consuming, untargeted distribution of genes, and high cost of cultivation. Therefore, it is suggested to develop novel techniques for the transportation of genes in crop plants using tailored nanoparticles (NPs) of manipulative and controlled high-performance features synthesized using green and chemical routes. It is observed that site-specific delivery of genes exhibits high efficacy in species-independent circumstances which leads to an increased level of productivity. Therefore, to achieve these outcomes, NPs can be utilized as gene nano-carriers for excellent delivery inside crops (i.e., cotton, tobacco, rice, wheat, okra, and maize) for desired genetic engineering modifications. As outcomes, this review provides an outline of the conventional techniques and current application of numerous nano-enabled gene delivery needed for crop gene manipulation, the benefits, and drawbacks associated with state-of-the-art techniques, which serve as a roadmap for the possible applicability of nanomaterials in plant genomic engineering as well as crop improvement in the future.

The presence of a selection marker in transgenic plants has raised public concerns regarding health safety. We have developed a CRISPR/Cas9‐based DNA delivery system termed transgenic selection‐associated fragment elimination (T‐SAFE). The T‐SAFE system comprises four cassettes: the selection marker, CRISPR/Cas9, spacer‐plus‐protospacer adjacent motif (SP), and the cargo. The first two cassettes, the selection marker and CRISPR/Cas9, are collectively referred to as SCC. The SCC is flanked by two identical SPs derived from the fruit fly Ebony gene, which efficiently facilitate the SCC cleavage and subsequently lead to self‐elimination of the selection marker upon integration of exogenous DNA into the plant genome. To inhibit the production of a functional Cas9 protein in bacteria, the IV2 intron of the potato ST‐LS1 gene has been incorporated into the Cas9 gene. Additionally, the Cas9 gene is driven by a reproductive cell‐specific or inducible promoter to avoid SCC cleavage in nonreproductive plant cells. These innovative features allow the T‐SAFE system to achieve an elimination efficiency of the selection marker ranging from 10%–30% in Arabidopsis and 5%–8% in rice, with a DNA delivery capacity of approximately 10 kb. This approach offers a safe means for genetically modifying plants.

Vaccination with DNA is one of the most promising novel immunization techniques against a variety of pathogens and tumors, for which conventional vaccination regimens have failed. DNA vaccines are able to stimulate both arms of the immune system simultaneously, without carrying the safety risks associated with live vaccines, therefore representing not only an alternative to conventional vaccines but also significant progress in the prevention and treatment of fatal diseases and infections. However, translation of the excellent results achieved in small animals to similar success in primates or large animals has so far proved to be a major hurdle. Moreover, biosafety issues, such as the removal of antibiotic resistance genes present in plasmid DNA used for vaccination, remain to be addressed adequately. This review describes strategies to improve the design and production of conventional plasmid DNA, including an overview of safety and regulatory issues. It further focuses on novel systems for the optimization of plasmid DNA and the development of diverse plasmid DNA delivery systems for vaccination purposes.

Polyamidoamine (PAMAM) dendrimers are one of the smallest and most precise nanomolecules available today, which have promising applications for the treatment of brain diseases. Each aspect of the dendrimer (core, size or generation, size of cavities, and surface functional groups) can be precisely modulated to yield a variety of nanocarriers for delivery of drugs and genes to brain cells in vitro or in vivo. Two of the most important criteria to consider when using PAMAM dendrimers for neuroscience applications is their safety profile and their potential to be prepared in a reproducible manner. Based on these criteria, features of PAMAM dendrimers are described to help the neuroscience researcher to judiciously choose the right type of dendrimer and the appropriate method for loading the drug to form a safe and effective delivery system to the brain.

DNA vaccines are ideal candidates for global vaccination purposes because they are inexpensive and easy to manufacture on a large scale such that even people living in low-income countries can benefit from vaccination. However, the potential of DNA vaccines has not been realized owing mainly to the poor cellular uptake of DNA in vivo resulting in the poor immunogenicity of DNA vaccines. In this review, we discuss the benefits and shortcomings of several promising and innovative non-biological methods of DNA delivery that can be used to increase cellular delivery and efficacy of DNA vaccines.

Bacterial vectors showcase promising capabilities for the delivery of nucleic acids, providing a versatile platform that stands as an alternative to traditional methods and viral vectors, offering enhanced safety profiles and broader cargo capacity.Developing attenuated bacterial vectors for nucleic acid involves intricate engineering to weaken pathogenicity while preserving immunogenic properties.Engineering approaches through designing genetic circuits involving vacuole escape and controlled lysis are paving the way for more precise and effective delivery of genetic material.Engineering bacterial conjugation and the Type 4 Secretion System presents an intriguing avenue for nucleic acid delivery.Future methods, such as nanoparticle-based delivery systems, are offering improved targeting and delivery mechanisms for therapeutic nucleic acids. The demand for diverse nucleic acid delivery vectors, driven by recent biotechnological breakthroughs, offers opportunities for continuous improvements in efficiency, safety, and delivery capacity. With their enhanced safety and substantial cargo capacity, bacterial vectors offer significant potential across a variety of applications. In this review, we explore methods to engineer bacteria for nucleic acid delivery, including strategies such as engineering attenuated strains, lysis circuits, and conjugation machinery. Moreover, we explore pioneering techniques, such as manipulating nanoparticle (NP) coatings and outer membrane vesicles (OMVs), representing the next frontier in bacterial vector engineering. We foresee these advancements in bacteria-mediated nucleic acid delivery, through combining bacterial pathogenesis with engineering biology techniques, as a pivotal step forward in the evolution of nucleic acid delivery technologies. The demand for diverse nucleic acid delivery vectors, driven by recent biotechnological breakthroughs, offers opportunities for continuous improvements in efficiency, safety, and delivery capacity. With their enhanced safety and substantial cargo capacity, bacterial vectors offer significant potential across a variety of applications. In this review, we explore methods to engineer bacteria for nucleic acid delivery, including strategies such as engineering attenuated strains, lysis circuits, and conjugation machinery. Moreover, we explore pioneering techniques, such as manipulating nanoparticle (NP) coatings and outer membrane vesicles (OMVs), representing the next frontier in bacterial vector engineering. We foresee these advancements in bacteria-mediated nucleic acid delivery, through combining bacterial pathogenesis with engineering biology techniques, as a pivotal step forward in the evolution of nucleic acid delivery technologies.

Despite its introduction more than three decades ago, gene therapy has fallen short of its expected potential for the treatment of a broad spectrum of diseases and continues to lack widespread clinical use. The fundamental limitation in clinical translatability of this therapeutic modality has always been an effective delivery system that circumvents degradation of the therapeutic nucleic acids, ensuring they reach the intended disease target. Plasmid DNA (pDNA) for the purpose of introducing exogenous genes presents an additional challenge due to its size and potential immunogenicity. Current pDNA methods include naked pDNA accompanied by electroporation or ultrasound, liposomes, other nanoparticles, and cell-penetrating peptides, to name a few. While the topic of numerous reviews, each of these methods has its own unique set of limitations, side effects, and efficacy concerns. In this review, we highlight emerging uses of exosomes for the delivery of pDNA for gene therapy. We specifically focus on bovine milk and colostrum-derived exosomes as a nano-delivery “platform”. Milk/colostrum represents an abundant, scalable, and cost-effective natural source of exosomes that can be loaded with nucleic acids for targeted delivery to a variety of tissue types in the body. These nanoparticles can be functionalized and loaded with pDNA for the exogenous expression of genes to target a wide variety of disease phenotypes, overcoming many of the limitations of current gene therapy delivery techniques.

Cell-penetrating peptides (CPPs) are widely used for the intracellular delivery of a variety of cargo molecules, including small molecules, peptides, nucleic acids, and proteins. Many cationic and amphiphilic CPPs have been developed; however, there have been few reports regarding hydrophobic CPPs. Herein, we have developed stapled hydrophobic CPPs based on the hydrophobic CPP, TP10, by introducing an aliphatic carbon side chain on the hydrophobic face of TP10. This side chain maintained the hydrophobicity of TP10 and enhanced the helicity and cell penetrating efficiency. We evaluated the preferred secondary structures, and the ability to deliver 5(6)-carboxyfluorescein (CF) as a model small molecule and plasmid DNA (pDNA) as a model nucleotide. The stapled peptide F-3 with CF, in which the stapling structure was introduced at Gly residues, formed a stable α-helical structure and the highest cell-membrane permeability via an endocytosis process. Meanwhile, peptide F-4 demonstrated remarkable stability when forming a complex with pDNA, making it the optimal choice for the efficient intracellular delivery of pDNA. The results showed that stapled hydrophobic CPPs were able to deliver small molecules and pDNA into cells, and that different stapling positions in hydrophobic CPPs can control the efficiency of the cargo delivery.