Genetic Editing Research Articles

The Evolution of Meganucleases Genome Engineering Tools: an overview

Human beings today have been able to pursue goals with non-random changes in the genome of living organisms. This is the process of genetic engineering or genome editing, which involves replacing, inserting, or deleting an arbitrary genomic sequence using an artificial restriction enzyme capable of cleaving a specific part of the genome. The ability to manipulate and modify genes and functional studies as well as awareness of the molecular basis of diseases and the development of new and targeted therapies with these techniques have been created. To date, different techniques have now been developed with the ability to edit targeted genomics including Zinc-finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALEN) and Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR associated protein 9 (Cas9) (CRISPR/Cas9). The fusing of a zinc-finger DNA-binding domain to a DNA-cleavage domain produces artificial restriction enzymes known as Zinc-finger nucleases (ZFNs). Zinc finger domains can be designed to target a specific DNA sequence which causes ZFNs to target unique sequences in complex genomes. TALENs are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain to cut DNA strands at specific locations. CRISPR/Cas9 is a bacterial immune system against viruses in which the Cas9 nucleus connects with a single-strand guide to a complementary target sequence and makes changes. In this study, we will review the evolution and various aspects of each one over time.

Injuries, Causes, & Treatments, IJVTPR 3(1): For “Such a Time as This”

This editorial presents the main questions for volume 3, issue 1 of the IJVTPR, titled Injuries, Causes, & Treatments. The focus is on the clinical outcomes still unfolding from billions of injections of the COVID-19 “vaccines” — the “synthetic gene therapies” administered, according to the Pharmaceutical Technology trackers in 2022, in more than 13 billion doses to more than 5 billion persons at the time of this writing. What are (1) the components in those injections that are (2) causing the observed clinical outcomes, and, (3) what treatments are possible for people who have received one or more of the injections?[1] Yuval Noah Harari has said that in a hundred years “the corona virus epidemic” will be marked “as the moment when a new regime of surveillance took over, especially, surveillance under the skin”. He says it enables overseers to “collect biometric data, analyze it, and understand people better than they understand themselves”. Or, as Klaus Schwab of the World Economic Forum put it, the power of “genetic editing” redefines everyone who receives it. Just so, the COVID era has elevated the subject-matter of this journal to a higher level than could even have been imagined in the summer of 2019 when it was being conceptualized. The Inaugural Issue was well underway before March 11, 2020 when the World Health Organization proclaimed the corona pandemic. The first issue of the journal would not appear until July 15, 2020, only months before what Schwab would call the “Fourth Industrial Revolution”. Is the “Fourth” one — with transformative gene editing at its core — an intended resuscitation of the “Third Reich”? Who could have imagined beforehand and who can now comprehend the events that are confronting us all?

Growth acceleration of Nile tilapia at 21 to 31 weeks of age with plasma-treated air-supplied water

This study aimed to develop a technique to accelerate fish growth without using genetic modification or genome editing. We have prepared a reactor with four pairs of opposed electrodes and a high-voltage power supply for the discharge. An arc discharge generates a plasma-treated gas in the reactor. Plasma-treated gas containing active species such as nitric oxide (NO) was generated via an arc discharge in the atmosphere and inserted into an aquarium containing Nile tilapia. No ozone was detected in the plasma-treated gas. Plasma treatment gas was supplied to the 20 L tank at a flow rate of 10 L per minute for varying supply times. The supply duration of plasma-treated air to the water tank was 0.5, 2, 5, and 15 min. Tanks were prepared for each of these four conditions, and gas was supplied daily at the same time. We observed that on supplying plasma-treated gas to tilapia from the 16th week of age for 5 min daily, the average length of the fish at 31 weeks of age was ∼1.5 times longer than that of the control fish. All other supply time conditions were also found to grow acceleration over the control. In the 15-minute supply time condition, individual differences in body length were more significant. A sample had more growth suppression than controls. In other words, the results suggest that an excess supply of active species can cause growth inhibition. These results suggest that an optimal supply of plasma-treated gas has a growth-promoting effect on fish. Key policy highlights The fish growth was accelerated by supplying plasma-treated air to the tank. The amount of ozone in the plasma-treated air was below the detection limit, and a large amount of RNS, such as nitric oxide, was generated. After an experimental period of 16 to 31 weeks, the average length of fish in the most significant growth condition was 1.5 times that of the control fish.

Electroactive Nanoinjection Platform for Intracellular Delivery and Genetic Editing

Electroactive Nanoinjection Platform for Intracellular Delivery and Genetic Editing

AI-POWERED PRECISION AGRICULTURE: RESHAPING FARMING FOR EFFICIENCY, SUSTAINABILITY, AND GLOBAL IMPACT

This article delves into the transformative effects of Artificial Intelligence (AI) on precision agriculture, also known as AgriTech or AgTech. The convergence of AI and farming practices reshapes the industry by leveraging machine learning, data analytics, and automation to optimize resource allocation and decision-making. Precision agriculture maximizes crop yields while minimizing waste through real-time data from AI technologies such as remote sensing, satellite imagery, and IoT sensors. AI-driven insights enable precise recommendations for irrigation, fertilization, and pest control, enhancing productivity and reducing environmental impact. Moreover, AI enhances crop health assessment, accelerates genetic research, and fosters climate-resilient crops. The article explores AI's potential in predictive analytics, genetic editing, global food security, and sustainable practices. Automation in agriculture tasks via AI-driven robotics and machinery elevates productivity. While AI offers positive environmental impact through resource optimization and energy efficiency, concerns about energy consumption and hardware management warrant attention. A balanced approach to AI's development, embracing energy-efficient algorithms and renewable energy integration, ensures its positive contribution to sustainable practices. The article concludes by discussing potential research directions and the complex interplay of AI's benefits and environmental concerns.

A dominant dpy-10 co-transformation marker using CRISPR/Cas9 and a linear repair template in Caenorhabditis tropicalis.

Caenorhabditis elegans is an excellent genetic model system with a large arsenal of forward and reverse genetic techniques. However, not all approaches are easily ported to related Caenorhabditis species (which are useful for gene conservation and gene pathway evolution studies). For CRISPR/Cas9 genetic editing, an easily screenable and dominant co-transformation marker is required - a secondary mutation that won't impact the phenotype of a desired mutation but is capable of being screened for in heterozygous mutants. We describe here the adaptation of a dominant dumpy/roller CRISPR/Cas9-induced mutation in the C. tropicalis dpy-10 orthologue.

Repensando o melhoramento de plantas e o sistemas de sementes na era de mudanças exponenciais

ABSTRACT The article stresses the need to reassess plant breeding and seed systems in our current era, characterized by rapidly shifting paradigms propelled by technological and scientific progress. It underscores the importance of incorporating cutting-edge technologies, fostering interdisciplinary collaboration, and implementing forward-looking policies to meet evolving societal and consumer demands while aligning with the Sustainable Development Goals (SDGs). Advancements in genetic editing, high-throughput technologies, precision breeding, and digital agriculture are transforming plant breeding, offering unprecedented capabilities to develop desirable traits, comprehend gene-environment interactions, and enhance crop efficiency. However, responsible innovation that considers ethical implications and regulatory frameworks remains essential in this context. Effective collaboration and engagement of stakeholders, including breeders, farmers, policymakers, and consumers, are vital for navigating the challenges and opportunities presented by this era of exponential change. Inclusive and multidisciplinary dialogues can harness diverse perspectives and experiences, contributing to a sustainable and resilient future for plant breeding and seed production. Ensuring access to diverse, high-quality seeds is crucial, especially in developing nations. Investments in seed infrastructure, capacity building, and farmer-centric approaches empower farmers and bolster seed security. In conclusion, the article underscores the significance of reimagining plant breeding and seed systems by embracing technology, promoting collaboration, and aligning strategies with the SDGs. By harnessing the potential of exponential changes, the envisioned future involves plant breeding and seed production that effectively address evolving societal and consumer needs, thereby contributing to agricultural resilience, global food security and sustainability.

Improving food security in India through non-conventional plant breeding technologies

Previous reviews addressed that there will be 170 crore people on the earth by 2050 the rise in the average income in emerging India. Hence, it will be the greatest challenge to meet food demand future. Aside from urbanization and land degradation, climate change also additional pressure on the food supply. These problems must be fundamentally changed because of their complexity and self-reinforcing nature. Crop improvement through breeding has been the main strategy for reducing poverty and increasing the food supply in India. New, improved crop varieties need to be developed for farmers to use to enhance food security. The biotechnological and non-conventional accelerated plant breeding methods that are not reliant on genetic engineering or gene editing are the main subject of this review. We focus particularly on the viability of short-term implementation by national agricultural research systems in underdeveloped nations. We contend that delaying the implementation of technology that can speed up reproduction is economically ineffective and supports the swift adoption of accelerated breeding techniques in the public sector. We recommend the employment of a technique called Rapid Generation Advance (RGA) as the most practical strategy for speed breeding in the public sector after taking into account a wide variety of considerations, including the economics of accelerated breeding.

The CRISPR/Cas System in Human Cancer.

The use of CRISPR as a genetic editing tool modified the oncology field from its basic to applied research for opening a simple, fast, and cheaper way to manipulate the genome. This chapter reviews some of the major uses of this technique for in vitro- and in vivo-based biological screenings, for cellular and animal model generation, and new derivative tools applied to cancer research. CRISPR has opened new frontiers increasing the knowledge about cancer, pointing to new solutions to overcome several challenges to better understand the disease and design better treatments.

The Unlikely Development of CAR T Cells: a Brief History and Prospects for the Future.

The quest to cure cancer has been one of the Holy Grails of medicine. The discovery I am going to share with you, CAR T cells, is a promising new form of therapy of cancer that offers the prospect of curing cancer using the immune system. CAR T cells are the first form of synthetic biology to enter the practice of medicine. The notion of using the immune system to fight cancer is an old idea. Over a century ago, bacteria were ground up and injected into patients with late-stage cancers, and occasionally the cancer would disappear. However, we now have precise tools for genetic editing and gene insertion like CRISPR/Cas9 to rewrite the DNA code, offering the possibility to improve the immune system over what has evolved in a Darwinian fashion. In 2017 for the first time, CAR T cells were approved for the treatment of cancer. Today they are used worldwide in more than 15,000 patients and they offer the promise to move beyond cancer to other fields of medicine such as autoimmune disease and heart disease. Here I will discuss the promises and challenges faced by the evolving CAR T cell industry.

A REVIEW ON RECENT ADVANCES IN ANIMAL BIOTECHNOLOGY

Animal biotechnology represents a cutting-edge field that has revolutionized our interactions with the animal kingdom. Recent advancements encompass various domains, including genetic editing techniques like CRISPR-Cas9, which allow precise genetic modifications for improved animal health and product quality. Cloning and reproductive technologies offer opportunities to preserve rare genetic traits and enhance livestock production, albeit accompanied by ethical and genetic diversity challenges. Transgenic animals, engineered with foreign genes, serve purposes from increased productivity to disease modeling, raising ethical and regulatory concerns. Disease resistance strategies involve selective breeding and gene editing to bolster animals’ immunity, promoting sustainable farming practices. Biopharming utilizes genetically modified animals for large-scale pharmaceutical production, balancing potential benefits with containment and safety issues. Conservation efforts employ advanced genetic and reproductive techniques to protect endangered species, necessitating careful ethical and ecological considerations. Animal biotechnology promises transformative possibilities but underscores the importance of responsible progress and ethical frameworks.

Romain Gary’s collaborative self-translations: Translational beyond translation

This article considers the possibility of translational processes beyond translation through a genetic editing approach to an understudied phenomenon in Translation Studies, namely the use of intermediary allograph translations in collaborative self-translation. It considers a self-translator’s practice of involving a hired translator to provide an initial translation of an entire work, later to be revised extensively by the author. With a focus on Romain Gary as its case study, it argues that an inductive extension of our notion of what is translational can offer a pathway to distinguishing between literal and metaphorical use of translation in literary theory. It thus suggests a potential alternative to existing translational discourse in interdisciplinary settings, as well as presenting a view of collaborative self-translation as a practice that can be fruitfully theorised within multiple paradigms in Translation Studies.

Molecular landscape and emerging therapeutic strategies in breast cancer brain metastasis.

Breast cancer (BC) is the most commonly diagnosed cancer worldwide. Advanced BC with brain metastasis (BM) is a major cause of mortality with no specific or effective treatment. Therefore, better knowledge of the cellular and molecular mechanisms underlying breast cancer brain metastasis (BCBM) is crucial for developing novel therapeutic strategies and improving clinical outcomes. In this review, we focused on the latest advances and discuss the contribution of the molecular subtype of BC, the brain microenvironment, exosomes, miRNAs/lncRNAs, and genetic background in BCBM. The blood-brain barrier and blood-tumor barrier create challenges to brain drug delivery, and we specifically review novel approaches to bypass these barriers. Furthermore, we discuss the potential application of immunotherapies and genetic editing techniques based on CRISPR/Cas9 technology in treating BCBM. Emerging techniques and research findings continuously shape our views of BCBM and contribute to improvements in precision therapies and clinical outcomes.

Gene editing for sickle cell disease and transfusion dependent thalassemias- A cure within reach

Sickle cell disease (SCD) is associated with significant morbidity and shortened life expectancy. Similarly, patients with transfusion dependent beta thalassemia (TdT) require life-long transfusion therapy, chelation therapy and significant organ dysfunction. Allogeneic transplantation from a matched family donor provided the only curative option for patients with SCD and TdT. Unfortunately, less than 20% of patients have a fully matched related donor and results using unrelated donor transplant were associated with high rate of complications. Ex vivo gene therapy through globin gene addition has been investigated extensively and recent encouraging preliminary data resulted in regulatory approval in patients with TdT. Recent improvements in our understanding of the molecular pathways controlling erythropoiesis and globin switching from fetal hemoglobin to adult hemoglobin offer a new and exciting therapeutic options. Rapid and substantial advances in genome editing tools using CRISPR/Cas9, have raised the possibility of genetic editing and correction in patient derived hematopoietic stem and progenitor cells. We will review results of gene editing approach that can induce fetal hemoglobin production in patients with SCD and TdT.

Preserving the integrity of genetic heritage in the face of new therapeutic options: bio-legal considerations on genetic editing

Preserving the integrity of genetic heritage in the face of new therapeutic options: bio-legal considerations on genetic editing

Single and multiplexed gene repression in solventogenic Clostridium via Cas12a-based CRISPR interference

The Gram-positive, spore-forming, obligate anaerobic firmicute species that make up the Clostridium genus have broad feedstock consumption capabilities and produce value-added metabolic products, but genetic manipulation is difficult, limiting their broad appeal. CRISPR-Cas systems have recently been applied to Clostridium species, primarily using Cas9 as a counterselection marker in conjunction with plasmid-based homologous recombination. CRISPR interference is a method that reduces gene expression of specific genes via precision targeting of a nuclease deficient Cas effector protein. Here, we develop a dCas12a-based CRISPR interference system for transcriptional gene repression in multiple mesophilic Clostridium species. We show the Francisella novicida Cas12a-based system has a broader applicability due to the low GC content in Clostridium species compared to CRISPR Cas systems derived from other bacteria. We demonstrate >99% reduction in transcript levels of targeted genes in Clostridium acetobutylicum and >75% reduction in Clostridium pasteurianum. We also demonstrate multiplexed repression via use of a single synthetic CRISPR array, achieving 99% reduction in targeted gene expression and elucidating a unique metabolic profile for their reduced expression. Overall, this work builds a foundation for high throughput genetic screens without genetic editing, a key limitation in current screening methods used in the Clostridium community.

Genetically edited T-cell membrane coated AIEgen nanoparticles effectively prevents glioblastoma recurrence

Glioblastoma stem cells (GSCs) are subpopulations of tumor-initiating cells responsible for glioblastoma (GBM) tumorigenesis and recurrence. Dual inhibition of vascular endothelium and GSCs is still a challenge due to their different pathological features. Here we present a combined all-in-control strategy to realize a local photothermal therapy (PTT). We designed T-cell-mimic nanoparticles with aggregation-induced emission (AIE) characteristics by coating the genetically engineered T cell membrane (CM) onto AIE nanoparticles (CM@AIE NPs). The CM shell was designed against CD133 and epidermal growth factor receptor (EGFR) which provides the possibility to target both GBM cells and GSCs for cancer therapy. CM@AIE NPs can serve as the tight junction (TJ) modulators to trigger an intracellular signaling cascade, causing TJ disruption and actin cytoskeleton reorganization to allow CM@AIE NPs to cross the blood-brain barrier (BBB) silently. The 980 nm excitation-triggered PTT can completely inhibit tumorigenesis and recurrence. The combination of CM-coating nanotechnology and genetic editing technique can inspire further development of synergetic techniques for preventing GBM recurrence.

Bacillus integrative plasmid system combining a synthetic gene circuit for efficient genetic modifications of undomesticated Bacillus strains

BackgroundOwing to CRISPR-Cas9 and derivative technologies, genetic studies on microorganisms have dramatically increased. However, the CRISPR-Cas9 system is still difficult to utilize in many wild-type Bacillus strains owing to Cas9 toxicity. Moreover, less toxic systems, such as cytosine base editors, generate unwanted off-target mutations that can interfere with the genetic studies of wild-type strains. Therefore, a convenient alternative system is required for genetic studies and genome engineering of wild-type Bacillus strains. Because wild-type Bacillus strains have poor transformation efficiencies, the new system should be based on broad-host-range plasmid-delivery systems.ResultsHere, we developed a Bacillus integrative plasmid system in which plasmids without the replication initiator protein gene (rep) of Bacillus are replicated in a donor Bacillus strain by Rep proteins provided in trans but not in Bacillus recipients. The plasmids were transferred to recipients through a modified integrative and conjugative element, which is a wide host range plasmid-delivery system. Genetic mutations were generated in recipients through homologous recombination between the transferred plasmid and the genome. The system was improved by adding a synthetic gene circuit for efficient screening of the desired mutations by double crossover recombination in recipient strains. The improved system exhibited a mutation efficiency of the target gene of approximately 100% in the tested wild-type Bacillus strains.ConclusionThe Bacillus integrative plasmid system developed in this study can generate target mutations with high efficiency when combined with a synthetic gene circuit in wild-type Bacillus strains. The system is free of toxicity and unwanted off-target mutations as it generates the desired mutations by traditional double crossover recombination. Therefore, our system could be a powerful tool for genetic studies and genome editing of Cas9-sensitive wild-type Bacillus strains.

Naturally selected CD7 CAR-T therapy without genetic editing demonstrates significant antitumour efficacy against relapsed and refractory acute myeloid leukaemia (R/R-AML)

BackgroundThe survival rate for patients with relapsed and refractory acute myeloid leukaemia (R/R-AML) remains poor, and treatment is challenging. Chimeric antigen receptor T cells (CAR-T cells) have been widely used for haematologic malignancies. Current CAR-T therapies for acute myeloid leukaemia mostly target myeloid-lineage antigens, such as CD123 and CD33, which may be associated with potential haematopoietic toxicity. As a lineage-specific receptor, CD7 is expressed in acute myeloid leukaemia cells and T cells but is not expressed in myeloid cells. Therefore, the use of CD7 CAR-T cells for R/R-AML needs to be further explored.MethodsIn this report, immunohistochemistry and flow cytometry were used to analyse CD7 expression in clinical samples from R/R-AML patients and healthy donors (HDs). We designed naturally selected CD7 CAR-T cells to analyse various functions and in vitro antileukaemic efficacy based on flow cytometry, and xenograft models were used to validate in vivo tumour dynamics.ResultsWe calculated the percentage of cells with CD7 expression in R/R-AML patients with minimal residual disease (MRD) (5/16, 31.25%) from our institution and assessed CD7 expression in myeloid and lymphoid lineage cells of R/R-AML patients, concluding that CD7 is expressed in T cells but not in myeloid cells. Subsequently, we designed and constructed naturally selected CD7 CAR-T cells (CD7 CAR). We did not perform CD7 antigen knockdown on CD7 CAR-T cells because CD7 molecule expression is naturally eliminated at Day 12 post transduction. We then evaluated the ability to target and kill CD7+ acute myeloid leukaemia cells in vitro and in vivo. Naturally selected CD7 CAR-T cells efficiently killed CD7+ acute myeloid leukaemia cells and CD7+ primary blasts of R/R-AML patients in vitro and significantly inhibited leukaemia cell growth in a xenograft mouse model.ConclusionNaturally selected CD7 CAR-T cells represent an effective treatment strategy for relapsed and refractory acute myeloid leukaemia patients in preclinical studies.

Consumer acceptance of new plant-breeding technologies: An application to the use of gene editing in fresh table grapes.

This study estimates consumers' willingness to pay for specific product (quality) and process (agronomic) attributes of table grapes, including taste, texture, external appearance, and the expected number of chemical applications, and for the breeding technology used to develop the plant. Considering varietal traits, on average our survey respondents were willing to pay the highest price premiums for specific offers of improvements in table grape taste and texture, followed by external appearance and expected number of chemical applications. Considering breeding methods, on average our respondents were willing to pay a small premium for table grapes developed using conventional breeding rather than gene editing (e.g., CRISPR). Results from a latent class model identify four different groups of consumers with distinct preferences for grape quality attributes and breeding technologies. The group of consumers most likely to reject gene editing considers both genetic engineering and gene editing to be breeding technologies that produce foods that are morally unacceptable and not safe to eat.