Genetic Engineering Research Articles

Microbial Perspectives on Polythene Biodegradation: Exploring the Role of Microorganisms in Addressing Plastic Pollution

Plastic pollution, particularly from polythene (polyethylene), has emerged as a significant environmental concern worldwide. In response to this challenge, microbial perspectives on polythene biodegradation have garnered attention as potential solutions to mitigate plastic pollution. This article provides an overview of the mechanisms underlying microbial polythene biodegradation, including surface erosion, biofilm formation, metabolic pathways, synergistic interactions, and adaptation. Furthermore, it explores the diversity of polythene-degrading microorganisms and their roles in plastic degradation across different environments. Environmental factors influencing polythene biodegradation, such as temperature, pH, moisture, and nutrient availability, are discussed, along with strategies to optimize degradation rates. Biotechnological approaches, including microbial consortia development and genetic engineering, are highlighted as promising avenues to enhance polythene degradation efficiency. The article concludes with a discussion on the potential of microbial perspectives to address plastic pollution and outlines future research directions in this field.

Internalization of transferrin-tagged Myxococcus xanthus encapsulins into mesenchymal stem cells

Currently, various functionalized nanocarrier systems are extensively studied for targeted delivery of drugs, peptides, and nucleic acids. Joining the approaches of genetic and chemical engineering may produce novel carriers for precise targeting different cellular proteins, which is important for both therapy and diagnosis of various pathologies. Here we present the novel nanocontainers based on vectorized genetically encoded Myxococcus xanthus (Mx) encapsulin, confining a fluorescent photoactivatable mCherry (PAmCherry) protein. The shells of such encapsulins were modified using chemical conjugation of human transferrin (Tf) prelabeled with a fluorescein-6 (FAM) maleimide acting as a vector. We demonstrate that the vectorized encapsulin specifically binds to transferrin receptors (TfRs) on the membranes of mesenchymal stromal/stem cells (MSCs) followed by internalization into cells. Two spectrally separated fluorescent signals from Tf-FAM and PAmCherry are clearly distinguishable and co-localized. It is shown that Tf-tagged Mx encapsulins are internalized by MSCs much more efficiently than by fibroblasts. It has been also found that unlabeled Tf effectively competes with the conjugated Mx-Tf-FAM formulations. That indicates the conjugate internalization into cells by Tf-TfR endocytosis pathway. The developed nanoplatform can be used as an alternative to conventional nanocarriers for targeted delivery of, e.g., genetic material to MSCs.

Molecular approaches to deploy singlet oxygen in a Leishmania model as an unassailable biocide for disease mitigation and vector control

Singlet oxygen (1O2) is a potent biocide potentially deployable for integrated control of tropical diseases and their insect vectors. This very short-lived free radical is highly destructive of cellular molecules when generated intracellularly. Most organisms, including parasites and vectors, are defenseless against 1O2 except for plants, which produce it abundantly during photosynthesis, hence, the acquisition of specific mechanisms for its detoxification. In the presence of O2 under physiological conditions, certain dyes or photosensitizers (PS), e.g., porphyrins and phthalocyanines (PC), are excitable by light to produce biocidal 1O2. Its half-life is in the order of microseconds, necessitating its intracellular generation in order to harness its biocidal activity most effectively. This is achievable by loading cells with PS for excitation with light to produce 1O2in situ. One example to achieve this is the genetic engineering of Leishmania to complement its inherent defects in porphyrin biosynthesis, resulting in cytosolic accumulation of abundant PS in the form of uroporphyrin 1 (URO). Another example is the chemical engineering of PC for hydrophilicity, thereby facilitating the endocytosis of such PS by cells. Leishmania loaded with cytosolic URO and endosomal PC are inactivated by the 1O2 produced via light-activation of these PS in the two different cell compartments. The inactivated Leishmania are nonviable, but have their natural vaccines and adjuvants well-preserved for prophylactic vaccination against experimental leishmaniasis. 1O2-inactivated Leishmania is potentially useful to serve as a platform for the safe and effective delivery of transgenically add-on vaccines against malignant and viral diseases in experimental models. Hydrophilic and cationic PC were also shown experimentally to act as a new type of dim light-activable insecticides, i.e., their mosquito larvicidal activities with <µM LD50 values. Similar results are expected by studying PC in additional laboratory insect models. A significant advantage has long been attributed to this type of insecticide, i.e., their aversion to a selection of genetic variants for resistance. An additional advantage of PC is their excitability to produce insecticidal 1O2 with deep-penetrating red or infrared light invisible to most insects, thereby potentially increasing the range and scope of targetable insect vectors.

Molecular Mechanisms of CBL-CIPK Signaling Pathway in Plant Abiotic Stress Tolerance and Hormone Crosstalk.

Abiotic stressors, including drought, salt, cold, and heat, profoundly impact plant growth and development, forcing elaborate cellular responses for adaptation and resilience. Among the crucial orchestrators of these responses is the CBL-CIPK pathway, comprising calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs). While CIPKs act as serine/threonine protein kinases, transmitting calcium signals, CBLs function as calcium sensors, influencing the plant's response to abiotic stress. This review explores the intricate interactions between the CBL-CIPK pathway and plant hormones such as ABA, auxin, ethylene, and jasmonic acid (JA). It highlights their role in fine-tuning stress responses for optimal survival and acclimatization. Building on previous studies that demonstrated the enhanced stress tolerance achieved by upregulating CBL and CIPK genes, we explore the regulatory mechanisms involving post-translational modifications and protein-protein interactions. Despite significant contributions from prior research, gaps persist in understanding the nuanced interplay between the CBL-CIPK system and plant hormone signaling under diverse abiotic stress conditions. In contrast to broader perspectives, our review focuses on the interaction of the pathway with crucial plant hormones and its implications for genetic engineering interventions to enhance crop stress resilience. This specialized perspective aims to contribute novel insights to advance our understanding of the potential of the CBL-CIPK pathway to mitigate crops' abiotic stress.

120 Gene editing: From the general public perspective

Abstract The public controversy around genetically engineered (GE) products, aka genetically modified organisms (GMO), has been ongoing for almost 40 yr fueled in part by well-funded misinformation campaigns disseminated by activist organizations. Polls reveal that public perception of GE animals is generally negative, with biomedical applications being more positively perceived than agricultural applications. Opposition to GE animals is often conflated with opposition to use of animals in research in general, as well as opposition to aspects of intensive animal agriculture. In general, concerns about animal biotechnology are influenced by views around the moral status of animals, the boundary between “natural” and “unnatural,” and perceived risks and benefits of GE animals to health and the environment (personal and cultural characteristics); the purpose of the application (e.g., disease resistance > fast growth), the method(s) being used, and the motivation of the research group making the genetic modification (public or private); and the species being modified (animal characteristics). There were three factors that aggravated opposition to GE products including 1) a lengthy and expensive regulatory step uniquely associated with commercializing products developed using that breeding method, 2) competing business interests (e.g., organic, non-GMO project) that were able to monetarize fear around the method to extract value (rent seeking) and selling their value-added (more expensive) product that avoided the use of GE, and 3) there is a mandatory labeling requirement to track/differentiate products produced with (or without) that breeding method to enable value-added marketing. Even so, when GE products that people wanted were allowed to reach the market (e.g., Impossible Burger with GE heme, GloFish, AquAdvantage salmon), they met with commercial acceptance. Now it has been suggested that the public will similarly reject animal products derived from gene edited (GnEd) animals, but that idea has only recently been put to the test as such food products were not previously available. A 2022 Japanese paper noted that with regard to GnEd food (gene knock-out tomatoes and fish) there was a “gap between anticipated and actual public response”. They noted that “even though there were some petitions against the use of genome-editing made by consumer groups; they did not develop into a mass mobilization, and media coverage was mostly positive.” These GnEd food products were developed by startups from Japanese universities, required no unique regulatory step before commercialization or mandatory labeling with Japanese regulatory agencies transparently sharing a summary of the information and notification on their websites, and businesses were encouraged to share information on products to respect The right of consumers to know. Successful commercialization of GnEd animal products has so far occurred in countries (e.g., Argentina, Brazil, Japan) where regulations treat simple GnEd products with no “foreign DNA” to be “non-GMO” and analogous to conventional breeding.

Application of Developmental Regulators for Enhancing Plant Regeneration and Genetic Transformation.

Establishing plant regeneration systems and efficient genetic transformation techniques plays a crucial role in plant functional genomics research and the development of new crop varieties. The inefficient methods of transformation and regeneration of recalcitrant species and the genetic dependence of the transformation process remain major obstacles. With the advancement of plant meristematic tissues and somatic embryogenesis research, several key regulatory genes, collectively known as developmental regulators, have been identified. In the field of plant genetic transformation, the application of developmental regulators has recently garnered significant interest. These regulators play important roles in plant growth and development, and when applied in plant genetic transformation, they can effectively enhance the induction and regeneration capabilities of plant meristematic tissues, thus providing important opportunities for improving genetic transformation efficiency. This review focuses on the introduction of several commonly used developmental regulators. By gaining an in-depth understanding of and applying these developmental regulators, it is possible to further enhance the efficiency and success rate of plant genetic transformation, providing strong support for plant breeding and genetic engineering research.

115 Gene editing: Where are we on campus and where can it go?

Abstract Editing the genome of livestock provides solutions to otherwise intractable problems and can provide a tool to better understand the role of specific proteins during development. The potential benefit for production agriculture is so great that the USDA Agricultural Research Service has invested in 4 positions now located in a research unit at the University of Missouri with a focus currently on swine and poultry. Genome editing can result in introducing alleles from divergent populations or novel alleles to produce animals that have new proteins, modified proteins or animals that no longer produce a protein. Applications of genome editing include improving disease resistance (viral and bacterial), improving digestion, disseminating valuable genetics, improved thermoregulation, changing the body/milk composition, and creating hypoallergenic meat. Changes in these animals will improve animal welfare, animal productivity and the sustainability of animal agriculture. In addition to improving animal agriculture, gene editing has enabled a new understanding of maternal recognition of pregnancy in the pig and a better understanding of molecules involved in growth and development of pigs. With new developments in editing of just somatic cells [somatic cell gene editing (SCGE), rather than germline editing], it will be possible to edit some cells in existing animals and thus change their phenotype. SCGE technologies such as this are being developed via the biomedical community as models to lead to human clinical applications. One such application is to correct cells in the lungs that produce the CFTR protein. Correction of just 10% of the cells in the lungs is estimated to correct the lung symptoms associated with cystic fibrosis. Similar applications in production agriculture might produce animals that have novel phenotypes of an altered carcass composition or better use of nutrients. Application of genetic engineering technologies such as genome editing is limited by biology and the imagination of the investigator.

From Gene Editing to Biofilm Busting: CRISPR-CAS9 Against Antibiotic Resistance-A Review.

In recent decades, the development of novel antimicrobials has significantly slowed due to the emergence of antimicrobial resistance (AMR), intensifying the global struggle against infectious diseases. Microbial populations worldwide rapidly develop resistance due to the widespread use of antibiotics, primarily targeting drug-resistant germs. A prominent manifestation of this resistance is the formation of biofilms, where bacteria create protective layers using signaling pathways such as quorum sensing. In response to this challenge, the CRISPR-Cas9 method has emerged as a ground-breaking strategy to counter biofilms. Initially identified as the "adaptive immune system" of bacteria, CRISPR-Cas9 has evolved into a state-of-the-art genetic engineering tool. Its exceptional precision in altering specific genes across diverse microorganisms positions it as a promising alternative for addressing antibiotic resistance by selectively modifying genes in diverse microorganisms. This comprehensive review concentrates on the historical background, discovery, developmental stages, and distinct components of CRISPR Cas9 technology. Emphasizing its role as a widely used genome engineering tool, the review explores how CRISPR Cas9 can significantly contribute to the targeted disruption of genes responsible for biofilm formation, highlighting its pivotal role in reshaping strategies to combat antibiotic resistance and mitigate the challenges posed by biofilm-associated infectious diseases.

Genetically Engineered CLDN18.2 CAR-T Cells Expressing Synthetic PD1/CD28 Fusion Receptors Produced Using a Lentiviral Vector.

This study aimed to develop synthetic Claudin18.2 (CLDN18.2) chimeric antigen receptor (CAR)-T (CAR-T) cells as a treatment for advanced gastric cancer using lentiviral vector genetic engineering technology that targets the CLDN18.2 antigen and simultaneously overcomes the immunosuppressive environment caused by programmed cell death protein 1 (PD-1). Synthetic CAR T cells are a promising approach in cancer immunotherapy but face many challenges in solid tumors. One of the major problems is immunosuppression caused by PD-1. CLDN18.2, a gastric-specific membrane protein, is considered a potential therapeutic target for gastric and other cancers. In our study, CLDN18.2 CAR was a second-generation CAR with inducible T-cell costimulatory (CD278), and CLDN18.2-PD1/CD28 CAR was a third-generation CAR, wherein the synthetic PD1/CD28 chimeric-switch receptor (CSR) was added to the second-generation CAR. In vitro, we detected the secretion levels of different cytokines and the killing ability of CAR-T cells. We found that the secretion of cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) secreted by three types of CAR-T cells was increased, and the killing ability against CLDN18.2-positive GC cells was enhanced. In vivo, we established a xenograft GC model and observed the antitumor effects and off-target toxicity of CAR-T cells. These results support that synthetic anti-CLDN18.2 CAR-T cells have antitumor effect and anti-CLDN18.2-PD1/CD28 CAR could provide a promising design strategy to improve the efficacy of CAR-T cells in advanced gastric cancer.

Genomic Advances in Biofortification of Iron and Zinc in Wheat (Triticum aestivum L.)

Majority of population that depend on wheat are found to be deficient in iron and zinc. About 20% of calories worldwide are derived from wheat. Milling of wheat reduces the bioavaibility of Fe and Zn in wheat grains. Micronutrient deficiency is the main cause of malnutrition worldwide and hidden hunger in underdeveloped countries. To overcome this problem it becomes necessary to develop varieties that are rich in iron and zinc content. Biofortification gets around these issues by enhancing the micronutrient content of wheat. The genetic approaches of biofortification particularly the non-conventional methods have proved to be the most efficient ones. These method includes QTL mapping approach, GWAS and transgenics. Although conventional methods for a long time has been used for developing varieties of wheat rich in Fe and Zn but conventional breeding for certain traits may be inconvenient and time-consuming and are prone to errors due to their reliance on traditional phenotyping or visual assessments. Several research have been undertaken to map QTLs of Fe and Zn concentration in wheat grains and have been used in elite genetic backgrounds to improve micronutrient content. Genomic studies revealed through GWAS exhibit high QTL resolution and allele coverage, which can be used for improvement of Fe and Zn content in wheat. Genetic engineering techniques or transgenics approaches allows genetic alteration of crops to improve the nutritional quality of wheat. Genomic approach of biofortification of wheat has proved to be the most efficient methods for increasing iron and zinc content in the crop.

CAR-T Cell Therapy in Pancreatic and Biliary Tract Cancers: An Updated Review of Clinical Trials.

Pancreatic and biliary tract cancers are digestive system tumors with dismal prognosis and limited treatment options. The effectiveness of conventional surgical interventions, radiation therapy, and systemic therapy is restricted in these cases. Furthermore, clinical trials have shown that immunotherapy using immune checkpoint inhibitors has only demonstrated modest clinical results when applied to patients with pancreatobiliary tumors. This highlights the importance of implementing combination immunotherapy approaches or exploring alternative therapeutic strategies to improve treatment outcomes. We reviewed the relevant literature on chimeric antigen receptor (CAR)-T cell therapy for pancreatobiliary cancers from PubMed/Medline and ClinicalTrials.gov and retrieved the relevant data accordingly. Attention was additionally given to the examination of grey literature with the aim of obtaining additional details regarding ongoing clinical trials. We mainly focused on abstracts and presentations and e-posters and slides of recent important annual meetings (namely ESMO Immuno-Oncology Congress, ESMO Congress, ASCO Virtual Scientific Program, ASCO Gastrointestinal Cancers Symposium). CAR-T cell therapy has emerged as a promising and evolving treatment approach for pancreatic and biliary tract cancer. This form of adoptive cell therapy utilizes genetic engineering to modify the expression of specific antibodies on the surface of T cells enabling them to target specific cancer-associated antigens and to induce potent anti-tumor activity. The aim of this review is to provide an updated summary of the available evidence from clinical trials that have explored the application of CAR-T cell therapy in treating pancreatobiliary cancers. While the utilization of CAR-T cell therapy in pancreatobiliary cancers is still in its initial phases with only a limited amount of clinical data available, the field is advancing rapidly, incorporating novel technologies to mitigate potential toxicities and enhance antigen-directed tumor eradication.

Polyhydroxyalkanoate Copolymer Production by Recombinant Ralstonia eutropha Strain 1F2 from Fructose or Carbon Dioxide as Sole Carbon Source

Ralstonia eutropha strain H16 is a chemoautotrophic bacterium that oxidizes hydrogen and accumulates poly[(R)-3-hydroxybutyrate] [P(3HB)], a prominent polyhydroxyalkanoate (PHA), within its cell. R. eutropha utilizes fructose or CO2 as its sole carbon source for this process. A PHA-negative mutant of strain H16, known as R. eutropha strain PHB−4, cannot produce PHA. Strain 1F2, derived from strain PHB−4, is a leucine analog-resistant mutant. Remarkably, the recombinant 1F2 strain exhibits the capacity to synthesize 3HB-based PHA copolymers containing 3-hydroxyvalerate (3HV) and 3-hydroxy-4-methyvalerate (3H4MV) comonomer units from fructose or CO2. This ability is conferred by the expression of a broad substrate-specific PHA synthase and tolerance to feedback inhibition of branched amino acids. However, the total amount of comonomer units incorporated into PHA was up to around 5 mol%. In this study, strain 1F2 underwent genetic engineering to augment the comonomer supply incorporated into PHA. This enhancement involved several modifications, including the additional expression of the broad substrate-specific 3-ketothiolase gene (bktB), the heterologous expression of the 2-ketoacid decarboxylase gene (kivd), and the phenylacetaldehyde dehydrogenase gene (padA). Furthermore, the genome of strain 1F2 was altered through the deletion of the 3-hydroxyacyl-CoA dehydrogenase gene (hbdH). The introduction of bktB-kivd-padA resulted in increased 3HV incorporation, reaching 13.9 mol% from fructose and 6.4 mol% from CO2. Additionally, the hbdH deletion resulted in the production of PHA copolymers containing (S)-3-hydroxy-2-methylpropionate (3H2MP). Interestingly, hbdH deletion increased the weight-average molecular weight of the PHA to over 3.0 × 106 on fructose. Thus, it demonstrates the positive effects of hbdH deletion on the copolymer composition and molecular weight of PHA.

Transcriptomics and starch biosynthesis analysis in leaves and developing seeds of mung bean provide a basis for genetic engineering of starch composition and seed quality.

Mung bean starch is distinguished by its exceptional high amylose content and regulation of starch biosynthesis in leaves and storage tissues, such as seeds, share considerable similarities. Genetic engineering of starch composition and content, requires detailed knowledge of starch biosynthetic gene expression and enzymatic regulation. In this study we applied detailed transcriptomic analyses to unravel the global differential gene expression patterns in mung bean leaves and in seeds during various stages of development. The objective was to identify candidate genes and regulatory mechanisms that may enable generation of desirable seed qualities through the use of genetic engineering. Notable differences in gene expression, in particular low expression of the Protein Targeting to Starch (PTST), starch synthase (SS) 3, and starch branching enzyme1 (SBE1) encoding genes in developing seeds as compared to leaves were evident. These differences were related to starch molecular structures and granule morphologies. Specifically, the starch molecular size distribution at different stages of seed development correlated with the starch biosynthesis gene expression of the SBE1, SS1, granule-bound starch synthases (GBSS) and isoamylase 1 (ISA1) encoding genes. Furthermore, putative hormonal and redox controlled regulation were observed, which may be explained by abscisic acid (ABA) and indole-3-acetic acid (IAA) induced signal transduction, and redox regulation of ferredoxins and thioredoxins, respectively. The morphology of starch granules in leaves and developing seeds were clearly distinguishable and could be correlated to differential expression of SS1. Here, we present a first comprehensive transcriptomic dataset of developing mung bean seeds, and combined these findings may enable generation of genetic engineering strategies of for example starch biosynthetic genes for increasing starch levels in seeds and constitute a valuable toolkit for improving mung bean seed quality.

Evaluating the intention to purchase genetically modified food by consumers in Panama

Genetically modified (GM) foods are important because of their ability to increase crop yields, resist disease and adverse weather conditions, and improve nutritional quality. However, their use generates controversy because of concerns about food safety, benefits and risks due to their consumption. It is therefore necessary to evaluate the importance of these variables. The current study evaluated the variables that explain consumers' intention to purchase GM food in Panama. It was carried out by prospective, quantitative, non-experimental, and correlational research of 385 consumers. A multivariate technique evaluated the model proposed in the current study. Attitude toward food safety showed a negative effect on perceived risks from GM food and a positive effect on perceived benefits from GM food. Also, Perceived risks from GM food presented a negative and significant effect on Attitudes towards GM food and Perceived benefits from GM food showed a positive and significant effect on Attitudes towards GM food. Finally, Attitudes towards GM food positively affected GM food purchase intention. Firms can use these outcomes to prepare new strategies for communication about their products with GM components. Also, governments can use the information to create education programs for the population about GM foods, benefits and risks. Future studies in other regions are needed.

Potential strategies for bioremediation of microplastic contaminated soil

The escalating production and ubiquitous presence of plastics and their degradation products, such as microplastics and nanoplastics, pose a significant environmental threat. Microplastics enter the soil through various pathways, including agricultural practices, plastic degradation, and wastewater disposal. Herein, we discussed the harmful effects of microplastics on the physicochemical properties of soil, plant growth, terrestrial fauna, and microbial activity, potentially affecting the stability and nutrient cycle of the soil ecosystem. This review delves into recent advances in potential microplastic bioremediation approaches, such as phytoremediation strategies utilized by plants and their associated microbes to accumulate, immobilize, and even degrade microplastics. Rhizosphere microorganisms play a crucial role in the degradation of microplastics, potentially utilizing them as a carbon source. Soil animals like earthworms, snails, and mealworms can also contribute significantly to bioremediation by ingesting and degrading microplastics through their gut microbiota. Various soil microorganisms, including bacteria and fungi, can degrade different microplastics with the help of enzymes such as laccase, esterase, peroxidase, oxidoreductase, and hydrolases and depolymerise the larger polymer chains into smaller units that ultimately mineralize them into CO2, H2O, and CH4. Genetic engineering and synthetic biology are also used to create strains with enhanced microplastic degrading and mineralization capabilities. It holds promise for efficient bioremediation but requires further research for real-world application and scalable implementation. Overall, this review comprehensively highlights the potential of bioremediation approaches and future recommendations for tackling microplastic pollution. Further research and development are crucial for enhancing biodegradation efficiency and scaling up this strategy for environmental protection.

Genetic engineering using CRISPR/Cas9 as a strategy for the treatment of Sickle Cell Anemia

Genetic engineering using CRISPR/Cas9 as a strategy for the treatment of Sickle Cell Anemia

Proficiency testing for event-specific quantification of genetically modified maize MON87427: Performance assessment based on the metrologically traceable reference values as assigned values

The Asia Pacific Metrology Program and the Accreditation Cooperation joint Proficiency Testing (PT) program for the quantification of genetically modified maize MON87427 was organized by the National Institute of Metrology, China, to enhance the measurement accuracy and metrological traceability in the region. Certified reference materials were employed as test samples; metrologically traceable certified reference values served as PT reference values (PTRVs) for evaluating the participants' results. The consensus values obtained from the participants were higher than the assigned values, potentially due to the systematic effects of DNA extraction process. The participants' relatively poor overall performance by the ζ-score compared with z-score demonstrates their need to thoroughly investigate quantification bias to elevate the measurement capability of genetically modified (GM) content and deepen their understanding of uncertainty estimation. This program confirmed the importance of using metrologically traceable reference values instead of consensus values as PTRV for reliable performance assessment.

Genome editing and kidney health.

Genome editing technologies, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas in particular, have revolutionized the field of genetic engineering, providing promising avenues for treating various genetic diseases. Chronic kidney disease (CKD), a significant health concern affecting millions of individuals worldwide, can arise from either monogenic or polygenic mutations. With recent advancements in genomic sequencing, valuable insights into disease-causing mutations can be obtained, allowing for the development of new treatments for these genetic disorders. CRISPR-based treatments have emerged as potential therapies, especially for monogenic diseases, offering the ability to correct mutations and eliminate disease phenotypes. Innovations in genome editing have led to enhanced efficiency, specificity and ease of use, surpassing earlier editing tools such as zinc-finger nucleases and transcription activator-like effector nucleases (TALENs). Two prominent advancements in CRISPR-based gene editing are prime editing and base editing. Prime editing allows precise and efficient genome modifications without inducing double-stranded DNA breaks (DSBs), while base editing enables targeted changes to individual nucleotides in both RNA and DNA, promising disease correction in the absence of DSBs. These technologies have the potential to treat genetic kidney diseases through specific correction of disease-causing mutations, such as somatic mutations in PKD1 and PKD2 for polycystic kidney disease; NPHS1, NPHS2 and TRPC6 for focal segmental glomerulosclerosis; COL4A3, COL4A4 and COL4A5 for Alport syndrome; SLC3A1 and SLC7A9 for cystinuria and even VHL for renal cell carcinoma. Apart from editing the DNA sequence, CRISPR-mediated epigenome editing offers a cost-effective method for targeted treatment providing new avenues for therapeutic development, given that epigenetic modifications are associated with the development of various kidney disorders. However, there are challenges to overcome, including developing efficient delivery methods, improving safety and reducing off-target effects. Efforts to improve CRISPR-Cas technologies involve optimizing delivery vectors, employing viral and non-viral approaches and minimizing immunogenicity. With research in animal models providing promising results in rescuing the expression of wild-type podocin in mouse models of nephrotic syndrome and successful clinical trials in the early stages of various disorders, including cancer immunotherapy, there is hope for successful translation of genome editing to kidney diseases.

Functional identification of DNA demethylase gene CaROS1 in pepper (Capsicum annuum L.) involved in salt stress.

Pepper, which is a widely cultivated important vegetable, is sensitive to salt stress, and the continuous intensification of soil salinization has affected pepper production worldwide. However, genes confer to salt tolerance are rarely been cloned in pepper. Since the REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylase that plays a crucial regulatory role in plants in response to various abiotic stresses, including salt stress. We cloned a ROS1 gene in pepper, named CaROS1 (LOC107843637). Bioinformatic analysis showed that the CaROS1 protein contains the HhH-GPD glycosylase and RRM_DME domains. qRT-PCR analyses showed that the CaROS1 was highly expressed in young and mature fruits of pepper and rapidly induced by salt stress. Functional characterization of the CaROS1 was performed by gene silencing in pepper and overexpressing in tobacco, revealed that the CaROS1 positively regulates salt tolerance ability. More detailly, CaROS1-silenced pepper were more sensitive to salt stress, and their ROS levels, relative conductivity, and malondialdehyde content were significantly higher in leaves than those of the control plants. Besides, CaROS1-overexpressing tobacco plants were more tolerant to salt stress, with a higher relative water content, total chlorophyll content, and antioxidant enzyme activity in leaves compared to those of WT plants during salt stress. These results revealed the CaROS1 dose play a role in salt stress response, providing the theoretical basis for salt tolerance genetic engineering breeding in pepper.

Deleting SUV39H1 in CAR-T cells epigenetically enhances the antitumor function.

SUV39H1 ablation in CAR-T cells epigenetically enhances the antitumor function (by Figdraw). (A) Schematic illustration of SUV39H1 ablation-mediated enhanced antitumor function of CAR-T cells. Functional CAR-T cells eventually transformed into dysfunctional exhausted CAR-T cells under the exposure of chronic tumor antigens, accompanied by reduced proliferation level, effector function, and stemness/memory characteristics, thereby limiting the antitumor activity so as to cause the recurrence of solid tumors. Upon genetic engineering of SUV39H1 ablation, SUV KO CAR-T cells are endowed with increased proliferation level and stemness/memory properties, accompanied by reduced effector/exhausted phenotype. Augmented SUV KO CAR-T cells after in vitro expansion intravenously infusion to mice achieved stronger and more persistent tumor rejection. (B) SUV39H1 ablation-mediated epigenetic reprogramming mechanism of CAR-T cells. Epigenetically, under the stimulation of chronic tumor antigens, exhausted CAR-T cells were characterized by downregulation of proliferation, effector and stemness/memory-associated genes and upregulation of exhaustion-associated genes. SUV39H1 genetic ablation increasedchromatin accessibility of stemness/memory-associated genes and reducedchromatin accessibility of inhibitory receptors and effector-associated genes in SUV KO CAR-T cells, epigenetically reprogramming human T cells to express higher levels of stemness/memory genes such as KLF2, LEF1 and TCF7 and lower levels of effector/exhaustion genes.