Genetic Editing Research Articles

An Anthocyanin-Based Visual Reporter System for Genetic Transformation and Genome Editing in Cassava

Cassava (Manihot esculenta Crantz) is a staple crop in tropical and subtropical regions, valued for its high starch content in roots. Effective genetic transformation and genome editing of cassava require efficient screening methods for transgenic and edited plants. In this study, a visual selection marker system using an R2R3-MYB transcription factor anthocyanin 1 gene (HbAN1, LOC110667474) from a rubber tree (Hevea brasiliensis Müll. Arg.) has been developed to facilitate the identification of transgenic cassava plants. Transgenic cassava lines expressing HbAN1 accumulated anthocyanins in their leaves, allowing for easy visual identification without the need for destructive assays or specialized equipment. Importantly, the accumulation of anthocyanins did not affect the regeneration or transformation efficiency of cassava. Additionally, the AR-CRISPR/Cas9-gRNA system with the HbAN1 gene as a marker produced MeCDD4 gene-edited cassava mutants with purple leaves, demonstrating successful editing. This anthocyanin-based visual reporter (AR) system will provide an effective tool for genetic transformation and genome editing in cassava.

Sonogenetics in the Treatment of Chronic Diseases: A New Method for Cell Regulation.

Sonogenetics is an innovative technology that integrates ultrasound with genetic editing to precisely modulate cellular activities in a non-invasive manner. This method entails introducing and activating mechanosensitive channels on the cell membrane of specific cells using gene delivery vectors. When exposed to ultrasound, these channels can be manipulated to open or close, thereby impacting cellular functions. Sonogenetics is currently being used extensively in the treatment of various chronic diseases, including Parkinson's disease, vision restoration, and cancer therapy. This paper provides a comprehensive review of key components of sonogenetics and focuses on evaluating its prospects and potential challenges in the treatment of chronic disease.

Advances in Soybean Genetic Improvement

The soybean (Glycine max) is a globally important crop due to its high protein and oil content, which serves as a key resource for human and animal nutrition, as well as bioenergy production. This review assesses recent advancements in soybean genetic improvement by conducting an extensive literature analysis focusing on enhancing resistance to biotic and abiotic stresses, improving nutritional profiles, and optimizing yield. We also describe the progress in breeding techniques, including traditional approaches, marker-assisted selection, and biotechnological innovations such as genetic engineering and genome editing. The development of transgenic soybean cultivars through Agrobacterium-mediated transformation and biolistic methods aims to introduce traits such as herbicide resistance, pest tolerance, and improved oil composition. However, challenges remain, particularly with respect to genotype recalcitrance to transformation, plant regeneration, and regulatory hurdles. In addition, we examined how wild soybean germplasm and polyploidy contribute to expanding genetic diversity as well as the influence of epigenetic processes and microbiome on stress tolerance. These genetic innovations are crucial for addressing the increasing global demand for soybeans, while mitigating the effects of climate change and environmental stressors. The integration of molecular breeding strategies with sustainable agricultural practices offers a pathway for developing more resilient and productive soybean varieties, thereby contributing to global food security and agricultural sustainability.

Unlocking Genome Editing: Advances and Obstacles in CRISPR/Cas Delivery Technologies

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats associated with protein 9) was first identified as a component of the bacterial adaptive immune system and subsequently engineered into a genome-editing tool. The key breakthrough in this field came with the realization that CRISPR/Cas9 could be used in mammalian cells to enable transformative genetic editing. This technology has since become a vital tool for various genetic manipulations, including gene knockouts, knock-in point mutations, and gene regulation at both transcriptional and post-transcriptional levels. CRISPR/Cas9 holds great potential in human medicine, particularly for curing genetic disorders. However, despite significant innovation and advancement in genome editing, the technology still possesses critical limitations, such as off-target effects, immunogenicity issues, ethical considerations, regulatory hurdles, and the need for efficient delivery methods. To overcome these obstacles, efforts have focused on creating more accurate and reliable Cas9 nucleases and exploring innovative delivery methods. Recently, functional biomaterials and synthetic carriers have shown great potential as effective delivery vehicles for CRISPR/Cas9 components. In this review, we attempt to provide a comprehensive survey of the existing CRISPR-Cas9 delivery strategies, including viral delivery, biomaterials-based delivery, synthetic carriers, and physical delivery techniques. We underscore the urgent need for effective delivery systems to fully unlock the power of CRISPR/Cas9 technology and realize a seamless transition from benchtop research to clinical applications.

Unlocking Innovation in Crop Resilience and Productivity: Breakthroughs in Biotechnology and Sustainable Farming

Recent advancements in agricultural biotechnology and sustainable practices are revolutionizing crop production and resilience, addressing global food security challenges. This review highlights cutting-edge innovations in plant sciences, including genetic engineering, genome editing technologies, and biotechnological interventions that enhance crop resistance to biotic and abiotic stresses. It discusses the integration of precision agriculture and digital farming tools that optimize resource use and boost productivity, providing data-driven insights for better crop management. Additionally, sustainable agricultural practices such as agroecology, organic farming, and integrated pest management are examined for their potential to reduce environmental impact while maintaining high yield. The review investigates the key practices like conservation tillage, crop rotation, and biological pest control, emphasizing their roles in enhancing soil health, biodiversity, and agricultural resilience. Significant contributions of biotechnological interventions, including genetic engineering and genome editing, are explored for developing stress-resistant and high-yield crop varieties. Moreover, the integration of precision agriculture and digital farming technologies is discussed for their potential to optimize resource utilization and improve crop management through advanced data analytics.

Novel Approaches in HIV-1 Cure: Genetic Editing and Gene Repression Techniques. Topic Review

Introduction: The worldwide challenge presented by human immunodeficiency virus type 1, affecting 39 million individuals, endures despite the highly active antiretroviral therapy. Limitations of it, including insufficiently addressing viral reservoirs leading to prolonged drug toxicity, resistance concerns, and economic burdens, have fueled exploration into alternative approaches. Gene editing and gene repression techniques have emerged as potential solutions. Objective: Conduct a comprehensive analysis of recent research following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, focusing on genetic modification and gene repression in HIV-1-infected samples. Methods: A meticulous search identified 413 articles, of which 76 met strict criteria. Inclusion criteria involved studies on gene editing and HIV-1 expression (2019–2023), emphasizing CCR5 repression. Exclusions targeted weak evidence, lacking practical efficacy, and non-relevance to HIV-1 or genetic tools. Duplicates and non-open-access articles were also excluded. Results: The identified strategies show promise in their ability to mitigate the chronicity of the infection. However, challenges such as off-target effects, ethical considerations surrounding in vivo, ex vivo, and germline editing, and variable efficacy across studies are evident. The imperative for continued evaluation, refinement of techniques, and the development of effective administration methods is underscored. Conclusions: This topic review provides a nuanced exploration of recent advancements in gene editing and gene repression techniques for managing HIV-1. While the findings suggest promising avenues, the identified challenges necessitate ongoing research and innovation in order to harness the full potential of these strategies in the quest for an effective HIV-1 solution.

Beyond the traditional distinctions of genome editing: evaluating a vulnerability framework

Over 40 years ago, the 1982 Splicing Life report outlined the two distinctions that have orientated much of the normative and legal landscape of genetic intervention or genome editing since – that of somatic versus germline (or heritable interventions) and medical versus non-medical (or enhancement) applications. During this time, these distinctions have been used to ethically prioritize some areas of research and potential application, such as somatic treatments, while considering others for prohibition, such as germline enhancements. Nevertheless, somatic interventions may also be done for controversial enhancement purposes while some germline interventions may be done with greater prima facie justification (e.g., the enhancement of athletic ability versus the avoidance of Tay-Sachs disease). Even with new somatic treatments that are generally lauded, exemplified with the case of Casgevy, many issues still arise – such as cost and access, particularly salient on a global level. The concerns over a dystopian future of genetic haves and have nots, as a result of enhancement and/or germline interventions, that perhaps may happen, should not distract us from a greater attention to what is happening in the here and now. In this paper, we will highlight the limits of the two distinctions in terms of moving from questions of “should a technology be used” to “how should a technology be used.” We argue that an additional focus on vulnerability and marginalization can be useful to support the attempt to better prioritize which interventions should be permitted or prohibited. We show how this can better dovetail with calls for effective (global) governance and reasonable consensus by focusing on the most urgent issues and developing policy accordingly, while leaving aside more abstract issues for further discussion.

Efficient genetic transformation and gene editing of Chinese cabbage using Agrobacterium rhizogenes.

A method using Agrobacterium rhizogenes-mediated callus production and plant regeneration enables efficient genetic transformation and gene editing in Chinese cabbage.

A Comparison of DNA-Methylation during Protoplast Culture of Ponkan Mandarin (Citrus reticulata Blanco) and Tobacco (Nicotiana tabacum L.)

The epigenetic variation in protoplast regeneration is a topic that has attracted interest recently. To elucidate the role of DNA methylation in the regeneration of protoplasts from the ponkan (Citrus reticulata), this study employs the methylation-sensitive amplification polymorphism (MSAP) molecular marker technique to analyze changes in DNA methylation levels and patterns during the isolation and culture of protoplasts from ponkan and tobacco. Additionally, differential DNA methylation fragments are cloned, sequenced, and subjected to bioinformatics analysis. The results reveal that, for non-regenerable ponkan mesophyll protoplasts, DNA methylation levels increase by 3.98% after isolation and then show a trend of initial decrease followed by an increase during culture. In contrast, for regenerable ponkan callus protoplasts and tobacco mesophyll protoplasts, DNA methylation levels decrease by 1.75% and 2.33%, respectively, after isolation. During culture, the DNA methylation levels of ponkan callus protoplasts first increase and then decrease, while those of tobacco mesophyll protoplasts show an opposite trend of initial decrease followed by an increase. Regarding DNA methylation patterns, ponkan mesophyll protoplasts exhibit primarily hypermethylation changes accompanied by a small amount of gene demethylation, whereas ponkan callus protoplasts are dominated by demethylation changes with some genes undergoing hypermethylation. The methylation exhibits dynamic changes in protoplast isolation regeneration. By recovering, cloning, sequencing, and performing BLASTn alignment analysis on specific methylation modification sites in the ponkan, 18 DNA sequences with high homology are identified which are found to be involved in various biological functions, thereby establishing a foundational basis for genetic editing in protoplasts.

Establishment of a Genetic Transformation and Gene Editing Method by Floral Dipping in Descurainia sophia

Establishment of a Genetic Transformation and Gene Editing Method by Floral Dipping in Descurainia sophia

Loss-of-function in testis-specific serine/threonine protein kinase triggers male infertility in an invasive moth

Genetic biocontrol technologies present promising and eco-friendly strategies for the management of pest and insect-transmitted diseases. Although considerable advancements achieve in gene drive applications targeting mosquitoes, endeavors to combat agricultural pests have been somewhat restricted. Here, we identify that the testis-specific serine/threonine kinases (TSSKs) family is uniquely expressed in the testes of Cydia pomonella, a prominent global invasive species. We further generated male moths with disrupted the expression of TSSKs and those with TSSKs disrupted using RNA interference and CRISPR/Cas9 genetic editing techniques, resulting in significant disruptions in spermiogenesis, decreased sperm motility, and hindered development of eggs. Further explorations into the underlying post-transcriptional regulatory mechanisms reveales the involvement of lnc117962 as a competing endogenous RNA (ceRNA) for miR-3960, thereby regulating TSSKs. Notably, orchard trials demonstrates that the release of male strains can effectively suppress population growth. Our findings indicate that targeting TSSKs could serve as a feasible avenue for managing C. pomonella populations, offering significant insights and potential strategies for controlling invasive pests through genetic sterile insect technique (gSIT) technology.

Efficient Method for Liposome-Based Gene Transfer in Cattle Embryos.

In vitro embryo production (IVP) in cattle is crucial for advancing genetic enhancement and preserving valuable genetic lineages, enabling precise genetic modifications and gene studies through modern techniques. Successful genetic manipulation in cattle embryos requires efficient delivery of exogenous DNA/RNA molecules. This research investigates the efficacy of a single embryo culture system for developing genetically modified zona-free (ZF) embryos and examines the use of liposome-based SAMTOR target siRNA transfer in these individually cultured ZF embryos. The findings indicated that the individual culture system resulted in increased cleavage rates, and blastocyst rates were minimally impacted. The new culture system effectively achieved SAMTOR silencing, with 8-16 cell embryos exhibiting reduction in transcript levels compared to control. Measurement of total protein content in the spent culture media was performed to validate the single-culture approach for further analytical applications. Total protein content analysis demonstrated the system's suitability for comprehensive evaluation of the embryo-media interaction, enhancing the scope for in-depth genetic research and applications. This research sheds light into an innovative method to improve genetic editing techniques in reproduction research.

Reactive Oxygen Species in Cystic Kidney Disease.

Polycystic kidney disease (PKD) is a rare but significant renal condition with major implications for global acute and chronic patient care. Oxidative stress and reactive oxygen species (ROS) can significantly alter its pathophysiology, clinical outcomes, and treatment, contributing to negative outcomes, including hypertension, chronic kidney disease, and kidney failure. Inflammation from ROS and existing cysts propagate the generation and accumulation of ROS, exacerbating kidney injury, pro-fibrotic signaling cascades, and interstitial fibrosis. Early identification and prevention of oxidative stress and ROS can contribute to reduced cystic kidney disease progression and improved longitudinal patient outcomes. Increased research regarding biomarkers, the pathophysiology of oxidative stress, and novel therapeutic interventions alongside the creation of comprehensive guidelines establishing methods of assessment, monitoring, and intervention for oxidative stress in cystic kidney disease patients is imperative to standardize clinical practice and improve patient outcomes. The integration of artificial intelligence (AI), genetic editing, and genome sequencing could further improve the early detection and management of cystic kidney disease and mitigate adverse patient outcomes. In this review, we aim to comprehensively assess the multifactorial role of ROS in cystic kidney disease, analyzing its pathophysiology, clinical outcomes, treatment interventions, clinical trials, animal models, and future directions for patient care.

Advancements in Lentil Genomics for Enhanced Crop Breeding: A Review

Lentil (Lens culinaris Medik) is an essential pulse crop that is widely grown for its high nutritional value, notably its high protein content, making it an important dietary component for vegetarians and vegans. Despite being the world's fifth most produced pulse, with large contributions from Canada and India, lentil production confronts obstacles such as poor productivity due to limited genetic improvement against biotic and abiotic stresses under rainfed cultivation conditions. Recent advances in lentil genetics and genomics, such as the discovery of genes related to yield, disease resistance, and nutritional content, have boosted breeding efforts to generate improved lentil varieties. The use of contemporary genomic techniques like molecular markers, marker-assisted selection (MAS), genomic selection (GS), and next-generation sequencing (NGS) technology has sped up the discovery of quantitative trait loci (QTLs) and the production of novel cultivars with superior agronomic characteristics. Databases such as NCBI and ENA, as well as specialized resources like KnowPulse, provide critical genomic data, while the creation of lentil genome assemblies, notably the CDC Redberry variety, has improved our understanding of lentil genetics. These resources help to solve the constraints of traditional breeding, particularly for complex characteristics impacted by genotype-environment interactions, opening the way for more robust and productive lentil varieties. Although the application of advanced tools such as genetic engineering, cisgenesis, and genome editing has moved more slowly in lentils than in other crops, their potential to improve lentil output is encouraging. Recent studies on lentil genomes, together with the creation of increased genetic resources and cutting-edge techniques, offer the ability to overcome production constraints and dramatically increase lentil production and quality throughout the world.

Decoding the genetic landscape of autism: A comprehensive review.

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition characterized by heterogeneous symptoms and genetic underpinnings. Recent advancements in genetic and epigenetic research have provided insights into the intricate mechanisms contributing to ASD, influencing both diagnosis and therapeutic strategies. To explore the genetic architecture of ASD, elucidate mechanistic insights into genetic mutations, and examine gene-environment interactions. A comprehensive systematic review was conducted, integrating findings from studies on genetic variations, epigenetic mechanisms (such as DNA methylation and histone modifications), and emerging technologies [including Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 and single-cell RNA sequencing]. Relevant articles were identified through systematic searches of databases such as PubMed and Google Scholar. Genetic studies have identified numerous risk genes and mutations associated with ASD, yet many cases remain unexplained by known factors, suggesting undiscovered genetic components. Mechanistic insights into how these genetic mutations impact neural development and brain connectivity are still evolving. Epigenetic modifications, particularly DNA methylation and non-coding RNAs, also play significant roles in ASD pathogenesis. Emerging technologies like CRISPR-Cas9 and advanced bioinformatics are advancing our understanding by enabling precise genetic editing and analysis of complex genomic data. Continued research into the genetic and epigenetic underpinnings of ASD is crucial for developing personalized and effective treatments. Collaborative efforts integrating multidisciplinary expertise and international collaborations are essential to address the complexity of ASD and translate genetic discoveries into clinical practice. Addressing unresolved questions and ethical considerations surrounding genetic research will pave the way for improved diagnostic tools and targeted therapies, ultimately enhancing outcomes for individuals affected by ASD.

Emerging CRISPR-Based Therapeutics for HIV Eradication

Human Immunodeficiency Virus (HIV) continued to pose a major global health challenge, with current antiretroviral therapies unable to fully eradicate the virus due to latent reservoirs and potential drug resistance. Emerging CRISPR-based gene editing technologies had the potential to revolutionize HIV treatment by targeting and disrupting the viral genome or modifying host cells to confer resistance. This article explored the mechanisms of CRISPR in HIV therapeutics, including excision of integrated HIV proviruses and genetic editing of host immune cells. It reviewed progress in CRISPR-based research through in vitro studies, animal models, and early clinical trials, highlighting both promising advancements and ongoing challenges such as delivery efficiency, off-target effects, and viral escape. The methodology employed in writing this review paper involved a comprehensive literature review and synthesis of recent research findings to assess the current state and future directions of CRISPR-based HIV therapies. Despite significant progress, challenges remained, and future research will need to focus on improving delivery systems, combining CRISPR with other therapies, and optimizing gene editing in stem cells to achieve long-term HIV eradication. Keywords: CRISPR-Cas9, HIV Therapy, Gene Editing, Latent Reservoirs, CCR5 Receptor.

Sustainable agricultural technologies of the future: Determination of adoption readiness for different consumer groups

The rapid increase in population and unsustainable agricultural practices has significantly damaged the environment. Our study explores how to achieve food security through environmentally friendly methods such as controlled environment agriculture (CEA), genetic editing (GE), and farming automation (FA), with an emphasis on the importance of consumer acceptance of these technologies. Analyzing data from 2138 Australians and 1760 New Zealanders, we employ a two-step clustering method—(1) hierarchical clustering and (2) k-means clustering—to categorize consumers on the basis of their demographic and lifestyle preferences into three segments that capture differences in attitudes toward new agricultural technologies: “Green Urbanites,” “Environmentally Unconcerned,” and “Skeptical Foodie.” Our findings reveal that Green Urbanites are the most open to adopting CEA, GE, and FA, while Skeptical Foodies and Environmentally Unconcerned are more reluctant. We contribute to theory by studying a novel facet of consumer acceptance of sustainable technologies and revealing how consumption, living, and lifestyle patterns motivate new technology acceptance in the agricultural sector. To inform practice, we suggest tailored strategies to increase engagement of the identified segments and promote wider acceptance of sustainable agricultural practices for enhanced food security.

Conquering Limitations: Exploring the Factors that Drive Successful Agrobacterium-Mediated Genetic Transformation of Recalcitrant Plant Species.

Agrobacterium-mediated transformation is a preferred method for genetic engineering and genome editing of plants due to its numerous advantages, although not all species exhibit transformability. Genetic engineering and plant genome editing methods are technically challenging in recalcitrant crop plants. Factors affecting the poor rate of transformation in such species include host genotype, Agrobacterium genotype, type of explant, physiological condition of the explant, vector, selectable marker, inoculation method, chemical additives, antioxidative compounds, transformation-enhancing compounds, medium formulation, optimization of culture conditions, and pre-treatments. This review provides novel insights into the key factors involved in gene transfer facilitated by Agrobacterium and proposes potential solutions to overcome existing barriers to transformation in recalcitrant species, thereby contributing to improvement programs for these species. This review introduces the key factors that impact the effectiveness of a molecular breeding program using Agrobacterium-mediated transformation, specifically focusing on recalcitrant plant species.

Rapid adaptive evolution of avian leukosis virus subgroup J in response to biotechnologically induced host resistance.

Genetic editing of the germline using CRISPR/Cas9 technology has made it possible to alter livestock traits, including the creation of resistance to viral diseases. However, virus adaptability could present a major obstacle in this effort. Recently, chickens resistant to avian leukosis virus subgroup J (ALV-J) were developed by deleting a single amino acid, W38, within the ALV-J receptor NHE1 using CRISPR/Cas9 genome editing. This resistance was confirmed both in vitro and in vivo. In vitro resistance of W38-/- chicken embryonic fibroblasts to all tested ALV-J strains was shown. To investigate the capacity of ALV-J for further adaptation, we used a retrovirus reporter-based assay to select adapted ALV-J variants. We assumed that adaptive mutations overcoming the cellular resistance would occur within the envelope protein. In accordance with this assumption, we isolated and sequenced numerous adapted virus variants and found within their envelope genes eight independent single nucleotide substitutions. To confirm the adaptive capacity of these substitutions, we introduced them into the original retrovirus reporter. All eight variants replicated effectively in W38-/- chicken embryonic fibroblasts in vitro while in vivo, W38-/- chickens were sensitive to tumor induction by two of the variants. Importantly, receptor alleles with more extensive modifications have remained resistant to the virus. These results demonstrate an important strategy in livestock genome engineering towards antivirus resistance and illustrate that cellular resistance induced by minor receptor modifications can be overcome by adapted virus variants. We conclude that more complex editing will be necessary to attain robust resistance.

Orange maker: a CRISPR/Cas9-mediated genome editing and screening project to generate orange-eyed DarkJedi GAL4 lines by undergraduate students

One of the greatest strengths of Drosophila genetics is its easily observable and selectable phenotypic markers. The mini-white marker has been widely used as a transgenic marker for Drosophila transgenesis. Flies carrying a mini-white construct can exhibit various eye colors ranging from pale orange to intense red, depending on the insertion site and gene dosage. Because the two copies of the mini-white marker show a stronger orange color, this is often used for selecting progenies carrying two transgenes together in a single chromosome after chromosomal recombination. However, some GAL4 lines available in the fly community originally have very strong red eyes. Without employing another marker, such as GFP, generating a recombinant chromosome with the strong red-eyed GAL4 and a desired UAS-transgene construct may be difficult. Therefore, we decided to change the red eyes of GAL4 lines to orange color. To change the eye color of the fly, we tested the CRISPR/Cas9 method with a guide RNA targeting the white gene with OK371-GAL4 and elav-GAL4. After a simple screening, we have successfully obtained multiple lines of orange-eyed OK371-GAL4 and elav-GAL4 that still maintain their original expression patterns. All of these simple experiments were performed by undergraduate students, allowing them to learn about a variety of different genetic experiments and genome editing while contributing to the fly research community by creating fruit fly lines that will be used in real-world research.