Genetic Modification Methods Research Articles

Assessment the Efficacy of the CRISPR System for Inducing Mutations in the AIMP2 Gene to Create a Cell Line Model of HLD17 Disease.

Hypomyelinating leukodystrophy-17 is a neurodevelopmental disorder caused by autosomal recessive mutations in the AIMP2 gene, resulting in a lack of myelin deposition during brain development, leading to variable neurological symptoms. Research on brain function in these disorders is challenging due to the lack of access to brain tissue. To overcome this problem, researchers have utilized different cell and animal models. The CRISPR-Cas9 system is considered the most optimal and effective method for genetic modification and developing cell models. We studied the efficacy of the CRISPR-Cas9 technology in inducing mutations in the AIMP2 gene in HEK293 cell lines. The study involved transfecting HEK293 cells with recombinant PX458 plasmids targeting spCas-9 and AIMP2 sgRNA. The cells were evaluated using fluorescent microscopy and enriched using serial dilution. The CRISPR/Cas9 plasmids were validated through PCR and Sanger sequencing. After serial dilution, AS-PCR, Sanger sequencing, and TIDE program analysis showed the construct successfully induces an indel mutation in HEK cells. Our findings demonstrated the great efficacy of the CRISPR system and produced a construct for inducing mutations in the AIMP2 gene, which can be utilized to edit the AIMP2 gene in nerve cells and create a cellular model of the HLD17 disease.

One-Step Genetic Modification by Embryonic Doral Aorta Injection of Adenoviral CRISPR/Cas9 Vector in Chicken.

In the avian species, genetic modification by cell nuclear transfer is infeasible due to its unique reproductive system. The in vitro primordial germ cell modification approach is difficult and cumbersome, although it is the main method of genetic modification in chickens. In the present study, the adenoviral CRISPR/Cas9 vector was directly microinjected into the dorsal aorta of chicken embryos to achieve in vivo genetic modification. The results demonstrated that keratin 75-like 4 (KRT75L4), a candidate gene crucial for feather development, was widely knocked out, and an 8bp deletion was the predominant mutation that occurred in multiple tissues in chimeras, particularly in the gonad (2.63-11.57%). As we expected, significant modification was detected in the sperm of G0 (0.16-4.85%), confirming the potential to generate homozygous chickens and establishing this vector as a simple and effective method for genetic modification in avian species.

CRISPR-based gene editing in plants: Focus on reagents and their delivery tools

Introduction: CRISPR-Cas9 technology has revolutionized plant genome editing, providing precise and efficient methods for genetic modification. This study focuses on the advancements and delivery of CRISPR-Cas9 in plant gene editing. Methods: A comprehensive search in scientific databases, including PubMed, ScienceDirect, and Google Scholar, was conducted to gather information on CRISPR-Cas9 gene editing and its delivery in precise gene modification in plants. Results: The evolving landscape of CRISPR nucleases has led to the development of innovative technologies, enhancing plant research. However, successful editing is contingent on efficient delivery of genome engineering reagents. CRISPR-based gene editing in plants utilizes diverse delivery methods: Agrobacterium-mediated transformation for bacterial transfer, biolistic transformation for physical gene insertion, electroporation for direct gene entry, expression of developmental regulators for gene expression modulation, and tobacco rattle virus as a viral vector, each offering distinct advantages for precise and efficient genetic modification in plants. Conclusion: CRISPR-Cas9 gene editing stands as a pivotal advancement in plant genetics, offering precise gene manipulation with applications in agriculture and biotechnology. The continuous refinement of reagent delivery tools reinforces CRISPR-Cas9's transformative role in plant genome editing, with significant implications for broader scientific applications.

Construction of peptide/plasmid DNA complexes for plant gene transfection via the basic leucine zipper domain

An important method for plant genetic modification is using peptide/pDNA complexes to transfer genes into plant cells. With conventional carrier peptides, the peptide sequence must contain a high amount of cationic amino acids to condense and introduce pDNA. As a result, the dissociation of pDNA from the complex is inefficient, often causing problems. Herein, we designed a new peptide carrier that mimics the basic leucine zipper (bZIP) domain of DNA-binding proteins, in which (LU)4 is the leucine zipper motif and (KUA)3 is the basic DNA-binding and cell-penetrating motif (U = α-aminoisobutyric acid). After (KUA)3-(LU)4 peptide was mixed with pDNA, DNA molecules were condensed to form nanoparticles of approximately 130 nm. Furthermore, when complexes of (KUA)3-(LU)4 peptide and pDNA were introduced into the leaves of Arabidopsis thaliana (A. thaliana), expression of the reporter protein was detected in the plant cells. Thus, (KUA)3-(LU)4 peptide that mimics the bZIP domain is a novel and efficient carrier for pDNA with high dissociation efficiency.

A Review on Citric Acid Production by Yarrowia lipolytica Yeast: Past and Present Challenges and Developments

The biosynthesis of citric acid (CA) and its derivatives is of great interest due to its wide range of applications in various manufacturing sectors. The fungus Aspergillus niger is mainly used for the commercial production of CA, using sucrose and molasses as the primary carbon sources. Since the 1960s, intensive research has been underway to introduce Yarrowia lipolytica yeast as an alternative to traditional fungal technology. This review discusses the practical uses of CA and its derivatives. Also, the challenges and developments that have led to efficient and green CA synthesis technologies using Y. lipolytica are outlined. The nutrient medium requirements and the use of various carbon sources, encompassing pure substrates and industry, agriculture, and food waste are considered. Additionally, the choice and improvement of strain producers, including efficient mutagenesis, genetic modification, and screening methods, are discussed.

Micropillars in Biomechanics: Role in Guiding Mesenchymal Stem Cells Differentiation and Bone Regeneration

AbstractEfficient bone repair relies on biomaterials that exhibit superior biocompatibility and enhanced osteogenic capabilities. In recent, micropatterning technology has emerged as a robust strategy for directing stem cell differentiation, offering a notable shift from traditional chemical induction or genetic modification methods by providing environmental cues to regulate cellular behavior. Among the various types of microstructures, micropillars have garnered significant attention due to their adjustable properties and high‐throughput experimental capabilities. These structures play a pivotal role in governing nuclear deformation and deciphering cellular responses to biomechanical cues. In this comprehensive review, the influence of micropillars on mesenchymal stem cells (MSCs), drawing upon two decades of research findings is critically assessed. The study comprehensively evaluates how micropillar substrates impact crucial cellular processes such as deformation, migration, adhesion, and ultimately, MSC fate determination toward an osteogenic lineage. By synthesizing and analyzing past studies, the potential regulatory mechanisms through which micropillars modulate MSC behavior are aimed to be elucidated. Furthermore, the utility of micropillars as a cutting‐edge biomechanical detection tool and platform for investigating cellular behaviors is explored. By projecting future applications, the review highlights the growing significance of micropillars in the dynamic field of biomechanics, underscoring their transformative potential in tissue engineering and regenerative medicine.

Plant genome modification technologies and some aspects of their practical use

Aim. To analyze modern methods of plant genome modification on the example of cereals and to evaluate the prospects of their use and practical significance. Methods. A review of available scientific sources and electronic resources containing data on existing and promising methods of genetic modification of cereals was conducted. Results. During the entire time of its development, mankind solved the issue of creating more productive agricultural crops that were adapted to the climatic conditions of existence. For this, domestication and selection based on spontaneous mutations were traditionally used. New technologies of genetic recombination, which have been developed in recent decades, make it possible to change the genome of plants in certain sites determined by the researcher. This has opened up unprecedented opportunities both in fundamental terms and in a whole range of applied aspects for elucidating the functions of specific genes and targeted improvement of the productivity of plants and some of them properties. Conclusions. Some modern new technologies of plant genome modifications are of significant scientific interest and practical importance. The use of these technologies is extremely important in many practical areas, especially for wheat and barley as the most agronomically important cereal crops in the world. Genome modifications agricultural crops require significant attention in terms of their safety and legal regulation of practical use. Information about modern methods of plant genome modification and directions for their use should be a mandatory element in educational courses on modern biotechnology in specialized educational institutions.

Complex Coacervates as a Promising Vehicle for mRNA Delivery: A Comprehensive Review of Recent Advances and Challenges.

Messenger RNA (mRNA)-based therapies have gained significant attention, following the successful deployment of mRNA-based COVID-19 vaccines. Compared with traditional methods of genetic modification, mRNA-based therapies offer several advantages, including a lower risk of genetic mutations, temporary and controlled therapeutic gene expression, and a shorter production time, which facilitates rapid responses to emerging health challenges. Moreover, mRNA-based therapies have shown immense potential in treating a wide range of diseases including cancers, immune diseases, and neurological disorders. However, the current limitations of non-viral vectors for efficient and safe delivery of mRNA therapies, such as low encapsulation efficiency, potential toxicity, and limited stability, necessitate the exploration of novel strategies to overcome these challenges and fully realize the potential of mRNA-based therapeutics. Coacervate-based delivery systems have recently emerged as promising strategies for enhancing mRNA delivery. Coacervates, which are formed by the aggregation of two or more macromolecules, have shown great potential in delivering a wide range of therapeutics due to their ability to form a separated macromolecular-rich fluid phase in an aqueous environment. This phase separation enables the entrapment and protection of therapeutic agents from degradation as well as efficient cellular uptake and controlled release. Additionally, the natural affinity of coacervates for mRNA molecules presents an excellent opportunity for enhancing mRNA delivery to targeted cells and tissues, making coacervate-based delivery systems an attractive option for mRNA-based therapies. This review highlights the limitations of current strategies for mRNA delivery and the advantages of coacervate-based delivery systems to enable mRNA therapeutics. Coacervates protect mRNA from enzymatic degradation and enhance cellular uptake, leading to sustained and controlled gene expression. Despite their promising properties, the specific use of coacervates as mRNA delivery vehicles remains underexplored. This review aims to provide a comprehensive overview of coacervate-mediated delivery of mRNA, exploring the properties and applications of different coacervating agents as well as the challenges and optimization strategies involved in mRNA encapsulation, release, stability, and translation via coacervate-mediated delivery. Through a comprehensive analysis of recent advancements and recommended future directions, our review sheds light on the promising role of coacervate-mediated delivery for RNA therapeutics, highlighting its potential to enable groundbreaking applications in drug delivery and gene therapy.

Molybdenum Nanofertilizer Boosts Biological Nitrogen Fixation and Yield of Soybean through Delaying Nodule Senescence and Nutrition Enhancement.

Soybean (Glycine max) is a crop of global significance and has low reliance on N fertilizers due to its biological nitrogen fixation (BNF) capacity, which harvests ambient N2 as a critical ecosystem service. BNF can be severely compromised by abiotic stresses. Enhancing BNF is increasingly important not only to alleviate global food insecurity but also to reduce the environmental impact of agriculture by decreasing chemical fertilizer inputs. However, this has proven challenging using current genetic modification or bacterial nodulation methods. Here, we demonstrate that a single application of a low dose (10 mg/kg) of molybdenum disulfide nanoparticles (MoS2 NPs) can enhance soybean BNF and grain yield by 30%, compared with conventional molybdate fertilizer. Unlike molybdate, MoS2 NPs can more sustainably release Mo, which then is effectively incorporated as a cofactor for the synthesis of nitrogenase and molybdenum-based enzymes that subsequently enhance BNF. Sulfur is also released sustainably and incorporated into biomolecule synthesis, particularly in thiol-containing antioxidants. The superior antioxidant enzyme activity of MoS2 NPs, together with the thiol compounds, protect the nodules from reactive oxygen species (ROS) damage, delay nodule aging, and maintain the BNF function for a longer term. The multifunctional nature of MoS2 NPs makes them a highly effective strategy to enhance plant tolerance to abiotic stresses. Given that the physicochemical properties of nanomaterials can be readily modulated, material performance (e.g., ROS capturing capacity) can be further enhanced by several synthesis strategies. This study thus demonstrates that nanotechnology can be an efficient and sustainable approach to enhancing BNF and crop yield under abiotic stress and combating global food insecurity.

Gestalt Principles Governed Fitness Function for Genetic Pythagorean Neutrosophic WASPAS Game Scene Generation

The maintenance of visual appeal and coherence in the procedural game scene generation is still a difficult problem. Traditional procedural game scene generation algorithms produce samples that show a noticeable resemblance to each other. The proposed algorithm allows us to add diverse game object compositions and increase creativity value in that way. Result diversity is formed by the proposed genetic algorithm modification and MCDM method based on the fitness function. Video game immersion is reached by aesthetic game element pattern composition, and one of the solutions for this issue is to apply automated aesthetic modelling of the generated game levels. In this research, the construction of fitness function was extended by the modelling of aesthetic principles, which were reverse-engineered from Gestalt principles. All rules were implemented by construction of a focal function with a square zone for each matrix cell of the single game scene. Five types of Gestalt rules were modelled and combined into a Pythagorean neutrosophic WASPAS method and the final score calculation algorithm was proposed. The proposed approach to generating game scenes strikes a balance between functionality and aesthetics to provide players with an engaging and immersive gaming experience.

Genetic improvement in Musa through modern biotechnological methods

Bananas, one of the most valued fruits worldwide, are produced in more than 135 countries in the tropics and subtropics for local consumption and export due to their tremendous nutritional value and ease of access. The genetic improvement of commercial crops is a crucial strategy for managing pests or other diseases and abiotic stress factors. Although conventional breeding has developed new hybrids with highly productive or agronomic performance characteristics, in some banana cultivars, due to the high level of sterility, the traditional breeding strategy is hampered. Therefore, modern biotechniques have been developed in a banana for genetic improvement. In vitro, culture techniques have been a basis for crop micropropagation for elite banana varieties and the generation of methods for genetic modification. This review includes topics of great interest for improving bananas and their products worldwide, from their origins to the different improvement alternatives. Keywords. Banana, genetic improvement, pest management, diseases, abiotic stress factors.

A minimally invasive fin scratching protocol for fast genotyping and early selection of zebrafish embryos

Current genetic modification and phenotyping methods in teleost fish allow detailed investigation of vertebrate mechanisms of development, modeling of specific aspects of human diseases and efficient testing of drugs at an organ/organismal level in an unparalleled fast and large-scale mode. Fish-based experimental approaches have boosted the in vivo verification and implementation of scientific advances, offering the quality guaranteed by animal models that ultimately benefit human health, and are not yet fully replaceable by even the most sophisticated in vitro alternatives. Thanks to highly efficient and constantly advancing genetic engineering as well as non-invasive phenotyping methods, the small zebrafish is quickly becoming a popular alternative to large animals’ experimentation. This approach is commonly associated to invasive procedures and increased burden. Here, we present a rapid and minimally invasive method to obtain sufficient genomic material from single zebrafish embryos by simple and precise tail fin scratching that can be robustly used for at least two rounds of genotyping already from embryos within 48 h of development. The described protocol betters currently available methods (such as fin clipping), by minimizing the relative animal distress associated with biopsy at later or adult stages. It allows early selection of embryos with desired genotypes for strategizing culturing or genotype–phenotype correlation experiments, resulting in a net reduction of “surplus” animals used for mutant line generation.

Engineered Exosomes for Tumor-Targeted Drug Delivery: A Focus on Genetic and Chemical Functionalization.

Cancer is the main cause of death worldwide. The limitations in traditional cancer therapies provoked the advance and use of several nanotechnologies for more effective and nontoxic cancer treatment. Along with synthetic nanocarriers, extracellular vesicles (EVs)-mediated drug delivery systems have aroused substantial interest. The term EVs refers to cell-derived nanovesicles, such as exosomes, with phospholipid-bound structures, participating in cell-to-cell communication. Exosomes are 30-150 nm vesicles that can transfer many biological molecules between cells. From a drug delivery standpoint, exosomes can be loaded with various therapeutic cargo, with the several advantages of low immunogenicity, high biocompatibility, transformative, and effective tumor targeting aptitude. The exosomal surface can be functionalized to improve tumor targeting ability of them. Researchers have genetically expressed or chemically linked various molecules on the surface of exosomes. Despite extensive investigation, clinical translation of exosome-based drug delivery remains challenging. In this review, we discuss various methods used to loading exosomes with therapeutic cargo. We describe examples of functionalized exosomes surface using genetic and chemical modification methods. Finally, this review attempts to provide future outlooks for exosome-based targeted drug delivery.

A Critical Review of Zebrafish Neurological Disease Models-2. Application: Functional and Neuroanatomical Phenotyping Strategies and Chemical Screens.

Extensive phylogenetic conservation of molecular pathways and neuroanatomical structures, associated with efficient methods for genetic modification, have been exploited increasingly to generate zebrafish models of human disease. A range of powerful approaches can be deployed to analyze these models with the ultimate goal of elucidating pathogenic mechanisms and accelerating efforts to find effective treatments. Unbiased neurobehavioral assays can provide readouts that parallel clinical abnormalities found in patients, although some of the most useful assays quantify responses that are not routinely evaluated clinically, and differences between zebrafish and human brains preclude expression of the full range of neurobehavioral abnormalities seen in disease. Imaging approaches that use fluorescent reporters and standardized brain atlases coupled with quantitative measurements of brain structure offer an unbiased means to link experimental manipulations to changes in neural architecture. Together, quantitative structural and functional analyses allow dissection of the cellular and physiological basis underlying neurological phenotypes. These approaches can be used as outputs in chemical modifier screens, which provide a major opportunity to exploit zebrafish models to identify small molecule modulators of pathophysiology that may be informative for understanding disease mechanisms and possible therapeutic approaches.

Investigating the Effect of Nudges on Consumers’ Willingness to Pay for Genetically Modified Corn Oil

Shifting from conventional methods of food production to genetic modification methods benefits sustainable agri-food production and environmental preservation. However, one of the main problems genetically modified food manufacturers have ever had to deal with is the public acceptability of GM foods. This study has two major objectives. First, it intends to apply principles from behavioral economics to investigate how consumers’ willingness to pay for GM corn oil can be affected. For this purpose, two different nudges are tested by providing consumers with positive information regarding GMO and changing the wording of the GMO label. Then, a comparison between the effectiveness of each of them is provided. Second, it investigates the impact of trust in GM food institutions, GMO information, and perceived GMO risk on both WTP for GM edible oil and the effectiveness of each nudge. A between-subjects choice experiment with a sample size of 550 Iranian corn oil consumers was conducted in Mashhad from March to April 2021. The results of mixed logit models indicate that both nudges affected consumer valuation of GM corn oil significantly, while their effectiveness differed according to the consumer level of trust in the GM food institutions and the perceived risk of GMO. Increasing consumer trust and information raises the WTP for GM corn oil; however, perceived risk has no effect. This study introduces effortless tools that GM food manufacturers can consider in their marketing strategies to affect consumers in the desired way.

Pig Coat Color Manipulation by MC1R Gene Editing.

Black coat color in pigs is determined by the dominant E allele at the MC1R locus. Through comparing MC1R gene sequences between recessive e and dominant ED1 alleles, we identified four missense mutations that could affect MC1R protein function for eumelanin synthesis. With the aim of devising a genetic modification method for pig coat color manipulation, we mutated the e allele in the Duroc breed to the dominant ED1 allele using CRISPR-mediated homologous recombination for the four mutation substitutions at the MC1R locus. The MC1R-modified Duroc pigs generated using the allele replacement strategy displayed uniform black coat color across the body. A genotyping assay showed that the MC1R-modified Duroc pigs had a heterozygous ED1/e allele at the MC1R locus; in addition, the pigs remained in the Duroc genetic background. Our work offers a gene editing method for pig coat color manipulation, which could value the culture of new pig varieties meeting the needs of diversified market.

Agrobacterium-mediated transformation – method of genetic modification of Triticum aestivum L. plants

Aim. Investigate the effect of Agrobacterium-mediated transformation in planta on seed tying and the frequency of transformation in winter wheat (Triticum aestivum L.). To analyze changes in the level of free L-proline (Pro) in transformed and control seedlings under normal / stress conditions and productivity indicators of biotechnological plants (T1) under normal growing conditions. Methods. Agrobacterium-mediated transformation in planta; PCR analysis, DNA electrophoresis; determination of seed tying frequency and transformation, Pro content, yield structure indicators. Results. Obtained transgenic wheat plants. The level of Pro in the tested variants under normal / stress conditions and indicators of T1 productivity of plants and their initial form under optimal water supply were studied. Conclusions. The susceptibility of the studied wheat genotypes to agrobacterial infection is shown. The frequency of seed tying after genetic transformation was 12.7 % and 5.4 % for plants of UK 106/19 and UK 171/19h, respectively. Transgenic seedlings had elevated levels of Pro. Complete incorporation of the vector construct was identified in 14 and 11 variants of genotypes UK 161/19 and UK 171/19h, respectively. Control and T1 biotechnological plants under normal growing conditions had similar yields.

Welfare considerations for farming black soldier flies, Hermetia illucens (Diptera: Stratiomyidae): a model for the insects as food and feed industry

Over two hundred billion black soldier flies (BSF,Hermetia illucens(Diptera: Stratiomyidae)) are reared annually across the globe, with the industry projected to grow substantially in the coming decade. Black soldier flies are being actively explored across the globe for use as livestock feed; fishmeal replacements; biodiesel; human, animal, and food waste management; and even sustainable human protein. Despite the huge number of individual insects reared and interest in BSF welfare by numerous producers and academics, there is no paper that considers the species-specific welfare of BSF in farmed conditions. We review factors that relate to BSF welfare in commercial rearing facilities, including: diseases/parasites, abiotic conditions (temperature, humidity/moisture, substrate aeration, light, pupation substrates, and adult spatial needs), adult and larval nutritional considerations, injury and crowding, handling-associated stress, selective breeding and genetic modification, environmental contaminants, and slaughter methods. We conclude with a discussion of the most pressing welfare concerns for the industry, recommendations for altering the conditions that give rise to them, and suggestions for future research directions that would lend valuable insights to BSF welfare. While this summary is BSF-centric, the core topic of animal welfare applies to all insect models currently, or in the future, produced as food and feed.

Construction of gene modification system with highly efficient and markerless for Monascus ruber M7.

Monascus spp. are traditional medicinal and edible filamentous fungi in China, and can produce various secondary metabolites, such as Monascus pigments (MPs) and citrinin (CIT). Genetic modification methods, such as gene knock-out, complementation, and overexpression, have been used extensively to investigate the function of related genes in Monascus spp.. However, the resistance selection genes that can have been used for genetic modification in Monascus spp. are limited, and the gene replacement frequency (GRF) is usually <5%. Therefore, we are committed to construct a highly efficient gene editing system without resistance selection marker gene. In this study, using M. ruber M7 as the starting strain, we successfully constructed a so-called markerlessly and highly genetic modification system including the mutants ΔmrpyrGΔmrlig4 and ΔmrpyrGΔmrlig4::mrpyrG, in which we used the endogenous gene mrpyrG from M. ruber M7 instead of the resistance marker gene as the screening marker, and simultaneously deleted mrlig4 related to non-homologous end joining in M. ruber M7. Then, the morphology, the growth rate, the production of MPs and CIT of the mutants were analyzed. And the results show that the mutant strains have normal mycelia, cleistothecia and conidia on PDA+Uridine(U) plate, the biomass of each mutant is also no different from M. ruber M7. However, the U addition also has a certain effect on the orange and red pigments yield of M. ruber M7, which needs our further study. Finally, we applied the system to delete multiple genes from M. ruber M7 separately or continuously without any resistance marker gene, and found that the average GRF of ΔmrpyrGΔmrlig4 was about 18 times of that of M. ruber M7. The markerlessly and highly genetic modification system constructed in current study not only will be used for multi-gene simultaneous modification in Monascus spp., and also lays a foundation for investigating the effects of multi-genes modification on Monascus spp..

Management of avian malaria in populations of high conservation concern

Avian malaria is a vector-borne disease that is caused by Plasmodium parasites. These parasites are transmitted via mosquito bites and can cause sickness or death in a wide variety of birds, including many threatened and endangered species. This Primer first provides contextual background for the avian malaria system including the life cycle, geographic distribution and spread. Then, we focus on recent advances in understanding avian malaria ecology, including how avian malaria can lead to large ecosystem changes and variation in host immune responses to Plasmodium infection. Finally, we review advances in avian malaria management in vulnerable bird populations including genetic modification methods suitable for limiting the effects of this disease in wild populations and the use of sterile insect techniques to reduce vector abundance.Graphical