Genetic Engineering Research Articles

Engineering Gut Symbionts: A Way to Promote Bee Growth?

Bees play a crucial role as pollinators, contributing significantly to ecosystems. However, the honeybee population faces challenges such as global warming, pesticide use, and pathogenic microorganisms. Promoting bee growth using several approaches is therefore crucial for maintaining their roles. To this end, the bacterial microbiota is well-known for its native role in supporting bee growth in several respects. Maximizing the capabilities of these microorganisms holds the theoretical potential to promote the growth of bees. Recent advancements have made it feasible to achieve this enhancement through the application of genetic engineering. In this review, we present the roles of gut symbionts in promoting bee growth and collectively summarize the engineering approaches that would be needed for future applications. Particularly, as the engineering of bee gut symbionts has not been advanced, the dominant gut symbiotic bacteria Snodgrassella alvi and Gilliamella apicola are the main focus of the paper, along with other dominant species. Moreover, we propose engineering strategies that will allow for the improvement in bee growth with listed gene targets for modification to further encourage the use of engineered gut symbionts to promote bee growth.

Picornavirus VP3 protein induces autophagy through the TP53-BAD-BAX axis to promote viral replication

ABSTRACT Macroautophagy/autophagy and apoptosis are pivotal interconnected host cell responses to viral infection, including picornaviruses. Here, the VP3 proteins of picornaviruses were determined to trigger autophagy, with the autophagic flux being triggered by the TP53-BAD-BAX axis. Using foot-and-mouth disease virus (FMDV) as a model system, we unraveled a novel mechanism of how picornavirus hijacks autophagy to bolster viral replication and enhance pathogenesis. FMDV infection induced both autophagy and apoptosis in vivo and in vitro. FMDV VP3 protein facilitated the phosphorylation and translocation of TP53 from the nucleus into the mitochondria, resulting in BAD-mediated apoptosis and BECN1-mediated autophagy. The amino acid Gly129 in VP3 is essential for its interaction with TP53, and crucial for induction of autophagy and apoptosis. VP3-induced autophagy and apoptosis are both essential for FMDV replication, while, autophagy plays a more important role in VP3-mediated pathogenesis. Mutation of Gly129 to Ala129 in VP3 abrogated the autophagic regulatory function of VP3, which significantly decreased the viral replication and pathogenesis of FMDV. This suggested that VP3-induced autophagy benefits viral replication and pathogenesis. Importantly, this Gly is conserved and showed a common function in various picornaviruses. This study provides insight for developing broad-spectrum antivirals and genetic engineering attenuated vaccines against picornaviruses. Abbreviations: 3-MA, 3-methyladenine; ATG, autophagy related; BAD, BCL2 associated agonist of cell death; BAK1, BCL2 antagonist/killer 1; BAX, BCL2 associated X, apoptosis regulator; BBC3/PUMA, BCL2 binding component 3; BCL2, BCL2 apoptosis regulator; BID, BH3 interacting domain death agonist; BIP-V5, BAX inhibitor peptide V5; CFLAR/FLIP, CASP8 and FADD like apoptosis regulator; CPE, cytopathic effects; CQ, chloroquine; CV, coxsackievirus; DAPK, death associated protein kinase; DRAM, DNA damage regulated autophagy modulator; EV71, enterovirus 71; FMDV, foot-and-mouth disease virus; HAV, hepatitis A virus; KD, knockdown; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MOI, multiplicity of infection; MTOR, mechanistic target of rapamycin kinase; PML, promyelocytic leukemia; PV, poliovirus; SVA, Seneca Valley virus; TCID50, 50% tissue culture infectious doses; TOR, target of rapamycin. TP53/p53, tumor protein p53; WCL, whole-cell lysate.

T-DNA insertion in Arabidopsis caused coexisting chromosomal inversion and duplication at megabase level

Agrobacteria-mediated transformation is widely used in plant genetic engineering to introduce exogenous genes and create mutant lines through random T-DNA insertion and gene disruption. When T-DNA fragments are inserted into the plant genome, it could cause chromosomal abnormalities. In this study, we investigated the genetic basis of pleiotropic phenotypes observed in the T-DNA insertion mutant lnc161. We discovered that there are four T-DNA insertions present in the lnc161 genome, which disrupted the genes LNC161 (AT3G05035), AT3G57400, AT5G05630, and AT5G16450, respectively. However, none of these insertions were the causative mutation that leads to the lnc161 phenotypes. Strikingly, through genetic analyses and high throughput sequencing, we found an inversion of about 19.8 Mb sequences between LNC161 and AT3G57400. Moreover, the sequences between AT5G05630 and AT5G16450 (about 3.7 Mb) were translocated from chromosome 5 to chromosome 3, adjacent to the inversion sequences, and were duplicated. This duplication led to an up-regulation of genes expression in this region, potentially resulting in pleiotropic morphological traits in lnc161. Overall, this study provides a case showing complex chromosomal re-arrangement induced by T-DNA insertion.

Targeted gene delivery systems for T-cell engineering.

T lymphocytes are indispensable for the host systems of defense against pathogens, tumors, and environmental threats. The therapeutic potential of harnessing the cytotoxic properties of T lymphocytes for antigen-specific cell elimination is both evident and efficacious. Genetically engineered T-cells, such as those employed in CAR-T and TCR-T cell therapies, have demonstrated significant clinical benefits in treating cancer and autoimmune disorders. However, the current landscape of T-cell genetic engineering is dominated by strategies that necessitate in vitro T-cell isolation and modification, which introduce complexity and prolong the development timeline of T-cell based immunotherapies. This review explores the complexities of gene delivery systems designed for T cells, covering both viral and nonviral vectors. Viral vectors are known for their high transduction efficiency, yet they face significant limitations, such as potential immunogenicity and the complexities involved in large-scale production. Nonviral vectors, conversely, offer a safer profile and the potential for scalable manufacturing, yet they often struggle with lower transduction efficiency. The pursuit of gene delivery systems that can achieve targeted gene transfer to T cell without the need for isolation represents a significant advancement in the field. This review assesses the design principles and current research progress of such systems, highlighting the potential for in vivo gene modification therapies that could revolutionize T-cell based treatments. By providing a comprehensive analysis of these systems, we aim to contribute valuable insights into the future development of T-cell immunotherapy.

Genetic engineering of a thermophilic acetogen, Moorella thermoacetica Y72, to enable acetoin production

Acetogens are among the key microorganisms involved in the bioproduction of commodity chemicals from diverse carbon resources, such as biomass and waste gas. Thermophilic acetogens are particularly attractive because fermentation at higher temperatures offers multiple advantages. However, the main target product is acetic acid. Therefore, it is necessary to reshape metabolism using genetic engineering to produce the desired chemicals with varied carbon lengths. Although such metabolic engineering has been hampered by the difficulty involved in genetic modification, a model thermophilic acetogen, M. thermoacetica ATCC 39073, is the case with a few successful cases of C2 and C3 compound production, other than acetate. This brief report attempts to expand the product spectrum to include C4 compounds by using strain Y72 of Moorella thermoacetica. Strain Y72 is a strain related to the type strain ATCC 39073 and has been reported to have a less stringent restriction-modification system, which could alleviate the cumbersome transformation process. A simplified procedure successfully introduced a key enzyme for acetoin (a C4 chemical) production, and the resulting strains produced acetoin from sugars and gaseous substrates. The culture profile revealed varied acetoin yields depending on the type of substrate and culture conditions, implying the need for further engineering in the future. Thus, the use of a user-friendly chassis could benefit the genetic engineering of M. thermoacetica.

Xenotransplantation of solid organs

Transplantation of genetically modified porcine hearts and kidneys could become asolution to the persistent shortage of human organ donors. Progress has been made in genetic engineering of donor pigs, preservation techniques after organ harvesting and immunosuppression using co-stimulation blockade with anti-CD40/CD40L monoclonal antibodies. Progress has also been made in in the development of methods that detect pathogenic porcine viruses and prevent their transmission to the recipient. As normal land breed pig organs continue to grow in the recipient to their original size, different pig breeds (such as Auckland Island pigs) are now used which reach a final size suitable for humans. Alternatively, aknock-out of the growth hormone receptor gene has been established, e.g., in the 10GM genetically modified pigs from Revivicor/United Therapeutics, USA. The first clinical pilot studies including patients suffering from terminal heart failure are expected to start in Germany in about 2years.

The isoprenyl chain length of coenzyme Q mediates the nutritional resistance of fungi to amoeba predation.

Ubiquinones (CoQ) are universal electron carriers in the respiratory chain of all aerobic bacteria and eukaryotes. Usually 8-10 isoprenyl units ensure their localization within the lipid bilayer. Members of the Saccharomyces clade among fungi are unique in using only 6. The reason for this is unclear. Here we provide evidence that the use of CoQ6 efficiently protects these fungi from predation by the ubiquitous fungivorous amoeba Protostelium aurantium which lacks its own biosynthetic pathway for this vitamin. The amoebae were starving on a diet of CoQ6 yeasts which could be complemented by either the addition of longer CoQs or the genetic engineering of a CoQ9 biosynthetic pathway.

The Effect of Short- and Long-Term Cryopreservation on Chicken Primordial Germ Cells

Primordial germ cells (PGCs) are the precursors of functional gametes and the only cell type capable of transmitting genetic and epigenetic information from generation to generation. These cells offer valuable starting material for cell-based genetic engineering and genetic preservation, as well as epigenetic studies. While chicken PGCs have demonstrated resilience in maintaining their germness characteristics during both culturing and cryopreservation, their handling remains a complex challenge requiring further refinement. Herein, the study aimed to compare the effects of different conditions (freezing-thawing and in vitro cultivation) on the expression of PGC-specific marker genes. Embryonic blood containing circulating PGCs was isolated from purebred Green-legged Partridgelike chicken embryos at 14–16 Hamburger–Hamilton (HH) embryonic development stage. The blood was pooled separately for males and females following sex determination. The conditions applied to the blood containing PGCs were as follows: (1) fresh isolation; (2) cryopreservation for a short term (2 days); and (3) in vitro culture (3 months) with long-term cryopreservation of purified PGCs (~2 years). To characterize PGCs, RNA isolation was carried out, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the expression levels of specific germ cell markers (SSEA1, CVH, and DAZL), as well as pluripotency markers (OCT4 and NANOG). The investigated genes exhibited consistent expression among PGCs maintained under diverse conditions, with no discernible differences observed between males and females. Notably, the analyzed markers demonstrated higher expression levels in PGCs when subjected to freezing than in their freshly isolated counterparts.

Biofortification of Plant- and Animal-Based Foods in Limiting the Problem of Microelement Deficiencies-A Narrative Review.

With a burgeoning global population, meeting the demand for increased food production presents challenges, particularly concerning mineral deficiencies in diets. Micronutrient shortages like iron, iodine, zinc, selenium, and magnesium carry severe health implications, especially in developing nations. Biofortification of plants and plant products emerges as a promising remedy to enhance micronutrient levels in food. Utilizing agronomic biofortification, conventional plant breeding, and genetic engineering yields raw materials with heightened micronutrient contents and improved bioavailability. A similar strategy extends to animal-derived foods by fortifying eggs, meat, and dairy products with micronutrients. Employing "dual" biofortification, utilizing previously enriched plant materials as a micronutrient source for livestock, proves an innovative solution. Amid biofortification research, conducting in vitro and in vivo experiments is essential to assess the bioactivity of micronutrients from enriched materials, emphasizing digestibility, bioavailability, and safety. Mineral deficiencies in human diets present a significant health challenge. Biofortification of plants and animal products emerges as a promising approach to alleviate micronutrient deficiencies, necessitating further research into the utilization of biofortified raw materials in the human diet, with a focus on bioavailability, digestibility, and safety.

Genetic Engineering of Filamentous Fungi: Prospects for Obtaining Fourth-Generation Biological Products

Filamentous fungi exhibit unparalleled potential as cell factories for protein production, owing to their adeptness in protein secretion and remarkable proficiency in post-translational modifications. This review delineates the role of filamentous fungi in bio-input technology across different generations and explores their capacity to generate secondary metabolites. Our investigation highlights filamentous fungi as frontrunners in the production of bioactive compounds, emphasizing the imperative nature of elucidating their metabolic repertoire. Furthermore, we delve into common strategies for genetic transformation in filamentous fungi, elucidating the underlying principles, advantages, and drawbacks of each technique. Taking a forward-looking approach, we explore the prospects of genome engineering, particularly the CRISPR-Cas9 technique, as a means to propel protein secretion in filamentous fungi. Detailed examination of the protein secretion pathways in these fungi provides insights into their industrial applications. Notably, extensive research within the scientific community has focused on Aspergillus and Trichoderma species for the industrial production of proteins and enzymes. This review also presents practical examples of genetic engineering strategies aimed at augmenting enzyme secretion in filamentous fungi for various industrial applications. These findings underscore the potential of filamentous fungi as versatile platforms for protein production and highlight avenues for future research and technological advancement in this field.

Analysis of the components of Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) and its regulation of γδ T-cell function

BackgroundMycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) is a peptide antigen released from the mycobacterial cytoplasm into the supernatant of Mycobacterium tuberculosis (Mtb) attenuated H37Ra strain after autoclaving at 121 °C for 20 min. Mtb-HAg can specifically induce γδ T-cell proliferation in vitro. However, the exact composition of Mtb-HAg and the protein antigens that are responsible for its function are currently unknown.MethodsMtb-HAg extracted from the Mtb H37Ra strain was subjected to LC‒MS mass spectrometry. Twelve of the identified protein fractions were recombinantly expressed in Escherichia coli by genetic engineering technology using pET-28a as a plasmid and purified by Ni–NTA agarose resin to stimulate peripheral blood mononuclear cells (PBMCs) from different healthy individuals. The proliferation of γδ T cells and major γδ T-cell subset types as well as the production of TNF-α and IFN-γ were determined by flow cytometry. Their proliferating γδ T cells were isolated and purified using MACS separation columns, and Mtb H37Ra-infected THP-1 was co-cultured with isolated and purified γδ T cells to quantify Mycobacterium viability by counting CFUs.ResultsIn this study, Mtb-HAg from the attenuated Mtb H37Ra strain was analysed by LC‒MS mass spectrometry, and a total of 564 proteins were identified. Analysis of the identified protein fractions revealed that the major protein components included heat shock proteins and Mtb-specific antigenic proteins. Recombinant expression of 10 of these proteins in by Escherichia coli genetic engineering technology was used to successfully stimulate PBMCs from different healthy individuals, but 2 of the proteins, EsxJ and EsxA, were not expressed. Flow cytometry results showed that, compared with the IL-2 control, HspX, GroEL1, and GroES specifically induced γδ T-cell expansion, with Vγ2δ2 T cells as the main subset, and the secretion of the antimicrobial cytokines TNF-α and IFN-γ. In contrast, HtpG, DnaK, GroEL2, HbhA, Mpt63, EsxB, and EsxN were unable to promote γδ T-cell proliferation and the secretion of TNF-α and IFN-γ. None of the above recombinant proteins were able to induce the secretion of TNF-α and IFN-γ by αβ T cells. In addition, TNF-α, IFN-γ-producing γδ T cells inhibit the growth of intracellular Mtb.ConclusionActivated γδ T cells induced by Mtb-HAg components HspX, GroES, GroEL1 to produce TNF-α, IFN-γ modulate macrophages to inhibit intracellular Mtb growth. These data lay the foundation for subsequent studies on the mechanism by which Mtb-HAg induces γδ T-cell proliferation in vitro, as well as the development of preventive and therapeutic vaccines and rapid diagnostic reagents.

Reviewing Soil Contaminant Remediation Techniques

Harmful contaminants generating from industry, agricultural and human activity are causing the degradation of soil health, along with detrimental effects on human and the environment. It is imperative to safeguard the soil from these dangerous pollutants by using soil remediation techniques that may be effective breakdown these dangerous toxins. A sustainable approach to remediate the soil from different contaminants is bioremediation. Bioremediation is a method where microbes are used to alleviate soil pollution effectively. Natural microorganisms like fungus, bacteria, and algae are employed in the bioremediation process to break down heavy metal (lead, arsenic, chromium etc.) or organic based. chemical contaminants. This study examines the use of microorganisms and various bioremediation methods, including genetic engineering, nanotechnology, and electro bioremediation, for recovering polluted soil. This investigation clarified the challenges associated with applying these bioremediation technologies and microorganisms, as well as their effects on the ecosystem and inherent soil microbial population. The goal of this current study is to illustrate the various technologies which are more effective in this remediation process.

Engineering the Signal Resolution of a Paper-Based Cell-Free Glutamine Biosensor with Genetic Engineering, Metabolic Engineering, and Process Optimization.

Specialized cancer treatments have the potential to exploit glutamine dependence to increase patient survival rates. Glutamine diagnostics capable of tracking a patient's response to treatment would enable a personalized treatment dosage to optimize the tradeoff between treatment success and dangerous side effects. Current clinical glutamine testing requires sophisticated and expensive lab-based tests, which are not broadly available on a frequent, individualized basis. To address the need for a low-cost, portable glutamine diagnostic, this work engineers a cell-free glutamine biosensor to overcome assay background and signal-to-noise limitations evident in previously reported studies. The findings from this work culminate in the development of a shelf-stable, paper-based, colorimetric glutamine test with a high signal strength and a high signal-to-background ratio for dramatically improved signal resolution. While the engineered glutamine test is important progress towards improving the management of cancer and other health conditions, this work also expands the assay development field of the promising cell-free biosensing platform, which can facilitate the low-cost detection of a broad variety of target molecules with high clinical value.

From trade-off to synergy: microbial insights into enhancing plant growth and immunity.

The reduction in crop yield caused by pathogens and pests presents a significant challenge to global food security. Genetic engineering, which aims to bolster plant defence mechanisms, emerges as a cost-effective solution for disease control. However, this approach often incurs a growth penalty, known as the growth-defence trade-off. The precise molecular mechanisms governing this phenomenon are still not completely understood, but they generally fall under two main hypotheses: a "passive" redistribution of metabolic resources, or an "active" regulatory choice to optimize plant fitness. Despite the knowledge gaps, considerable practical endeavours are in the process of disentangling growth from defence. The plant microbiome, encompassing both above- and below-ground components, plays a pivotal role in fostering plant growth and resilience to stresses. There is increasing evidence which indicates that plants maintain intimate associations with diverse, specifically selected microbial communities. Meta-analyses have unveiled well-coordinated, two-way communications between plant shoots and roots, showcasing the capacity of plants to actively manage their microbiota for balancing growth with immunity, especially in response to pathogen incursions. This review centers on successes in making use of specific root-associated microbes to mitigate the growth-defence trade-off, emphasizing pivotal advancements in unravelling the mechanisms behind plant growth and defence. These findings illuminate promising avenues for future research and practical applications.

Modernizing and harmonizing regulatory data requirements for genetically modified crops-perspectives from a workshop.

Genetically modified (GM) crops that have been engineered to express transgenes have been in commercial use since 1995 and are annually grown on 200 million hectares globally. These crops have provided documented benefits to food security, rural economies, and the environment, with no substantiated case of food, feed, or environmental harm attributable to cultivation or consumption. Despite this extensive history of advantages and safety, the level of regulatory scrutiny has continually increased, placing undue burdens on regulators, developers, and society, while reinforcing consumer distrust of the technology. CropLife International held a workshop at the 16th International Society of Biosafety Research (ISBR) Symposium to examine the scientific basis for modernizing global regulatory frameworks for GM crops. Participants represented a spectrum of global stakeholders, including academic researchers, GM crop developers, regulatory consultants, and regulators. Concurrently examining the considerations of food and feed safety, along with environmental safety, for GM crops, the workshop presented recommendations for a core set of data that should always be considered, and supplementary (i.e., conditional) data that would be warranted only on a case-by-case basis to address specific plausible hypotheses of harm. Then, using a case-study involving a hypothetical GM maize event expressing two familiar traits (insect protection and herbicide tolerance), participants were asked to consider these recommendations and discuss if any additional data might be warranted to support a science-based risk assessment or for regulatory decision-making. The discussions during the workshop highlighted that the set of data to address the food, feed, and environmental safety of the hypothetical GM maize, in relation to a conventional comparator, could be modernized compared to current global regulatory requirements. If these scientific approaches to modernize data packages for GM crop regulation were adopted globally, GM crops could be commercialized in a more timely manner, thereby enabling development of more diverse GM traits to benefit growers, consumers, and the environment.

CRISPR-Cas9 delivery strategies and applications: Review and update.

Nowadays, a significant part of the investigations carried out in the medical field belong to cancer treatment. Generally, conventional cancer treatments, including chemotherapy, radiotherapy, and surgery, which have been used for a long time, are not sufficient, especially in malignant cancers. Because genetic mutations cause cancers, researchers are trying to treat these diseases using genetic engineering tools. One of them is clustered regularly interspaced short palindromic repeats (CRISPR), a powerful tool in genetic engineering in the last decade. CRISPR, which forms the CRISPR-Cas structure with its endonuclease protein, Cas, is known as a part of the immune system (adaptive immunity) in bacteria and archaea. Among the types of Cas proteins, Cas9 endonuclease has been used in many scientific studies due to its high accuracy and efficiency. This review reviews the CRISPR system, focusing on the history, classification, delivery methods, applications, new generations, and challenges of CRISPR-Cas9 technology.

Designing molecules: directing stem cell differentiation.

Stem cells have been widely applied in regenerative and therapeutic medicine for their unique regenerative properties. Although much research has shown their potential, it remains tricky in directing stem cell differentiation. The advancement of genetic and therapeutic technologies, however, has facilitated this issue through development of design molecules. These molecules are designed to overcome the drawbacks previously faced, such as unexpected differentiation outcomes and insufficient migration of endogenous or exogenous MSCs. Here, we introduced aptamer, bacteriophage, and biological vectors as design molecules and described their characteristics. The methods of designing/developing discussed include various Systematic Evolution of Ligands by Exponential Enrichment (SELEX) procedures, in silico approaches, and non-SELEX methods for aptamers, and genetic engineering methods such as homologous recombination, Bacteriophage Recombineering of Electroporated DNA (BRED), Bacteriophage Recombineering with Infectious Particles (BRIP), and genome rebooting for bacteriophage. For biological vectors, methods such as alternate splicing, multiple promoters, internal ribosomal entry site, CRISPR-Cas9 system and Cre recombinase mediated recombination were used to design viral vectors, while non-viral vectors like exosomes are generated through parental cell-based direct engineering. Besides that, we also discussed the pros and cons, and applications of each design molecule in directing stem cell differentiation to illustrate their great potential in stem cells research. Finally, we highlighted some safety and efficacy concerns to be considered for future studies.

Screening and genetic engineering of marine-derived Aspergillus terreus for high-efficient production of lovastatin

BackgroundLovastatin has widespread applications thanks to its multiple pharmacological effects. Fermentation by filamentous fungi represents the major way of lovastatin production. However, the current lovastatin productivity by fungal fermentation is limited and needs to be improved.ResultsIn this study, the lovastatin-producing strains of Aspergillus terreus from marine environment were screened, and their lovastatin productions were further improved by genetic engineering. Five strains of A. terreus were isolated from various marine environments. Their secondary metabolites were profiled by metabolomics analysis using Ultra Performance Liquid Chromatography–Mass spectrometry (UPLC–MS) with Global Natural Products Social Molecular Networking (GNPS), revealing that the production of secondary metabolites was variable among different strains. Remarkably, the strain of A. terreus MJ106 could principally biosynthesize the target drug lovastatin, which was confirmed by High Performance Liquid Chromatography (HPLC) and gene expression analysis. By one-factor experiment, lactose was found to be the best carbon source for A. terreus MJ106 to produce lovastatin. To improve the lovastatin titer in A. terreus MJ106, genetic engineering was applied to this strain. Firstly, a series of strong promoters was identified by transcriptomic and green fluorescent protein reporter analysis. Then, three selected strong promoters were used to overexpress the transcription factor gene lovE encoding the major transactivator for lov gene cluster expression. The results revealed that compared to A. terreus MJ106, all lovE over-expression mutants exhibited significantly more production of lovastatin and higher gene expression. One of them, LovE-b19, showed the highest lovastatin productivity at a titer of 1512 mg/L, which represents the highest production level reported in A. terreus.ConclusionOur data suggested that combination of strain screen and genetic engineering represents a powerful tool for improving the productivity of fungal secondary metabolites, which could be adopted for large-scale production of lovastatin in marine-derived A. terreus.

Making the leap from technique to treatment - genetic engineering is paving the way for more efficient phage therapy.

Bacteriophages (phages) are viruses specific to bacteria that target them with great efficiency and specificity. Phages were first studied for their antibacterial potential in the early twentieth century; however, their use was largely eclipsed by the popularity of antibiotics. Given the surge of antimicrobial-resistant strains worldwide, there has been a renaissance in harnessing phages as therapeutics once more. One of the key advantages of phages is their amenability to modification, allowing the generation of numerous derivatives optimised for specific functions depending on the modification. These enhanced derivatives could display higher infectivity, expanded host range or greater affinity to human tissues, where some bacterial species exert their pathogenesis. Despite this, there has been a noticeable discrepancy between the generation of derivatives in vitro and their clinical application in vivo. In most instances, phage therapy is only used on a compassionate-use basis, where all other treatment options have been exhausted. A lack of clinical trials and numerous regulatory hurdles hamper the progress of phage therapy and in turn, the engineered variants, in becoming widely used in the clinic. In this review, we outline the various types of modifications enacted upon phages and how these modifications contribute to their enhanced bactericidal function compared with wild-type phages. We also discuss the nascent progress of genetically modified phages in clinical trials along with the current issues these are confronted with, to validate it as a therapy in the clinic.

Spatially Precise Genetic Engineering at the Electrode-Tissue Interface.

The interface between electrodes and neural tissues plays a pivotal role in determining the efficacy and fidelity of neural activity recording and modulation. While considerable efforts have been made to improve the electrode-tissue interface, the majority of studies have primarily concentrated on the development of biocompatible neural electrodes through abiotic materials and structural engineering. In this study, an approach is presented that seamlessly integrates abiotic and biotic engineering principles into the electrode-tissue interface. Specifically, ultraflexible neural electrodes with short hairpin RNAs (shRNAs) designed to silence the expression of endogenous genes within neural tissues are combined. The system facilitates shRNA-mediated knockdown of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and polypyrimidine tract-binding protein 1 (PTBP1), two essential genes associated in neural survival/growth and neurogenesis, within specific cell populations located at the electrode-tissue interface. Additionally, it is demonstrated that the downregulation of PTEN in neurons can result in an enlargement of neuronal cell bodies at the electrode-tissue interface. Furthermore, the system enables long-term monitoring of neuronal activities following PTEN knockdown in a mouse model of Parkinson's disease and traumatic brain injury. The system provides a versatile approach for genetically engineering the electrode-tissue interface with unparalleled precision, paving the way for the development of regenerative electronics and next-generation brain-machine interfaces.