Zinc Finger Nucleases Research Articles

Effect of PI3-kinase inhibitors on DNA double strand break repair pathways: observations using a site specific DSB induction system.

We established two types of site-specific DNA double strand breaks (DSB) induction systems to elucidate factors which affect the efficiency or quality of DSB repair. For mutation assays, a site specific DSB was generated by the transient expression of a zinc finger nuclease which targets the human HPRT1 gene. A cell line in which time and site specific DSBs can be generated in a HR reporter construct was used for homology-directed repair (HR) analysis. By using these two systems, we investigated the effects of PI3-kinase inhibitors on the efficiency and quality of DSB repair. Ataxia telangiectasia mutated (ATM) kinase inhibition resulted a decrease in mutant frequency and a slight increase in deletion-type mutations accompanied by microhomology. Furthermore, the HR frequency increased significantly when ATM kinase activity was inhibited. Thus, ATM kinase activity might be involved in the suppression of DSB end resection, and this may promote DSB repair through canonical non-homologous end joining.

CRISPR-Cas9: A Cutting-edge Genome-editing Technology with Many Potential Applications

Unregulated human activities are responsible for climate change which is a major factor for the emergence of new infectious (bacterial, viral, fungus, and parasitic) and non-infectious diseases (genetic disorders, cancers, etc.). To tackle this situation, we must have better therapeutics, diagnostics, and vaccines for the treatment and prevention of emerging diseases in humans and animals. To address the aforementioned issues, CRISPR-Cas9, a revolutionary genome editing tool, can help us develop better therapeutics, diagnostics, and vaccines in a short time. In addition, it can be used to achieve food security by improving productivity, adaptability, and resilience traits in plants and animals. In nature, most bacteria and archaea have CRISPR as a part of their adaptive immune system that helps bacteria defend themselves from phages, viruses, and other foreign genetic elements. CRISPR-Cas9 is a highly effective, simple, and accurate genome editing tool compared to previous genome editing tools such as meganucleases, Zinc finger nucleases (ZFNs), and transcription activators like effector nucleases (TALENs). This review article discusses the different components and working of the CRISPR-Cas9 system, and how CRISPR-Cas9 plays a significant role in the development of next-generation therapeutics, diagnostics, vaccine platforms, and improved crops and livestock species for food security.

Zinc finger nuclease-mediated gene editing in hematopoietic stem cells results in reactivation of fetal hemoglobin in sickle cell disease

BIVV003 is a gene-edited autologous cell therapy in clinical development for the potential treatment of sickle cell disease (SCD). Hematopoietic stem cells (HSC) are genetically modified with mRNA encoding zinc finger nucleases (ZFN) that target and disrupt a specific regulatory GATAA motif in the BCL11A erythroid enhancer to reactivate fetal hemoglobin (HbF). We characterized ZFN-edited HSC from healthy donors and donors with SCD. Results of preclinical studies show that ZFN-mediated editing is highly efficient, with enriched biallelic editing and high frequency of on-target indels, producing HSC capable of long-term multilineage engraftment in vivo, and express HbF in erythroid progeny. Interim results from the Phase 1/2 PRECIZN-1 study demonstrated that BIVV003 was well-tolerated in seven participants with SCD, of whom five of the six with more than 3 months of follow-up displayed increased total hemoglobin and HbF, and no severe vaso-occlusive crises. Our data suggest BIVV003 represents a compelling and novel cell therapy for the potential treatment of SCD.

Precision Genome Editing Techniques in Gene Therapy: Current State and Future Prospects.

Precision genome editing is a rapidly evolving field in gene therapy, allowing for the precise modification of genetic material. The CRISPR and Cas systems, particularly the CRISPRCas9 system, have revolutionized genetic research and therapeutic development by enabling precise changes like single-nucleotide substitutions, insertions, and deletions. This technology has the potential to correct disease-causing mutations at their source, allowing for the treatment of various genetic diseases. Programmable nucleases like CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs) can be used to restore normal gene function, paving the way for novel therapeutic interventions. However, challenges, such as off-target effects, unintended modifications, and ethical concerns surrounding germline editing, require careful consideration and mitigation strategies. Researchers are exploring innovative solutions, such as enhanced nucleases, refined delivery methods, and improved bioinformatics tools for predicting and minimizing off-target effects. The prospects of precision genome editing in gene therapy are promising, with continued research and innovation expected to refine existing techniques and uncover new therapeutic applications.

Ethical aspects associated with genome alteration techniques applied to animal reproduction research.

The revolution in biology triggered by the different genome-editing tools has of course arrived to the research field of animal reproduction. Yeast meganucleases, zinc-finger nucleases, TALEN and, particularly, the several generations of CRISPR tools have landed in animal reproduction thereby providing novel strategies to optimize or modify some of the features and capabilities of the recipient animals. All these genome-editing proposals and activities are associated with ethical considerations regarding how those planned genome alterations might affect important animal welfare issues. The ethical dimension of all these genome editing must be seriously considered. Hence, all ethical aspects bound to any given genome-edited allele in animals should be discussed in order to ensure that we are maximizing benefits and reducing any potential risk or negative considerations of these modifications. In this review, I will summarize some of the experiments reported aiming to investigate or improve animal reproduction and I will address the ethics issues that should also be considered.

Direct protein delivery into intact Arabidopsis cells for genome engineering

Intracellular delivery of biomolecules is a prerequisite for genetic techniques such as recombinant engineering and genome editing. Realizing the full potential of this technology requires the development of safe and effective methods for delivering protein tools into cells. In this study, we demonstrated the spontaneous internalization of exogenous proteins into intact cells and root tissue of whole plants of Arabidopsis thaliana. We termed this internalization phenomenon as protein Delivery Independent of Vehicles or Equipment (DIVE), which efficiently delivered genome engineering proteins including Cre recombinase and zinc-finger nucleases (ZFN) into plant cells. Using protein DIVE, we achieved less toxic protein delivery than electroporation with up to 94% efficiency in Arabidopsis cell culture and 19% genome modification in Arabidopsis plants that was maintained in regenerated tissue. This work illustrates the potential of protein DIVE for a wide range of applications, including genome engineering in plants.

Traditional Strategies and Cutting-Edge Technologies Used for Plant Disease Management: A Comprehensive Overview

Agriculture plays a fundamental role in ensuring global food security, yet plant diseases remain a significant threat to crop production. Traditional methods to manage plant diseases have been extensively used, but they face significant drawbacks, such as environmental pollution, health risks and pathogen resistance. Similarly, biopesticides are eco-friendly, but are limited by their specificity and stability issues. This has led to the exploration of novel biotechnological approaches, such as the development of synthetic proteins, which aim to mitigate these drawbacks by offering more targeted and sustainable solutions. Similarly, recent advances in genome editing techniques—such as meganucleases (MegNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)—are precise approaches in disease management, but are limited by technical challenges and regulatory concerns. In this realm, nanotechnology has emerged as a promising frontier that offers novel solutions for plant disease management. This review examines the role of nanoparticles (NPs), including organic NPs, inorganic NPs, polymeric NPs and carbon NPs, in enhancing disease resistance and improving pesticide delivery, and gives an overview of the current state of nanotechnology in managing plant diseases, including its advantages, practical applications and obstacles that must be overcome to fully harness its potential. By understanding these aspects, we can better appreciate the transformative impact of nanotechnology on modern agriculture and can develop sustainable and effective strategies to mitigate plant diseases, ensuring enhanced agricultural productivity.

Therapeutic Gene Editing for Hemoglobinopathies.

In the last ten years, a consistent number of clinical studies have evaluated different gene approaches for the treatment of patients with sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT). Initial studies of gene therapy for hemoglobinopathies involved the use of lentiviral vectors to add functional copies of the gene encoding β-globin in defective CD34 cells; more recently, gene editing techniques have been used involving either CRISPR-Cas9, transcription activation-like effector protein nuclease, zinc finger nuclease, and base editing to either induce fetal hemoglobin production at therapeutic levels or to genetically repair the underlying molecular defect causing the disease. Here, we review recent gene editing studies that have started the development of a new era in the treatment of hemoglobinopathies and, in general, monoallelic hereditary diseases.

Editing microbes to mitigate enteric methane emissions in livestock.

Livestock production significantly contributes to greenhouse gas (GHG) emissions particularly methane (CH4) emissions thereby influencing climate change. To address this issue further, it is crucial to establish strategies that simultaneously increase ruminant productivity while minimizing GHG emissions, particularly from cattle, sheep, and goats. Recent advancements have revealed the potential for modulating the rumen microbial ecosystem through genetic selection to reduce methane (CH4) production, and by microbial genome editing including CRISPR/Cas9, TALENs (Transcription Activator-Like Effector Nucleases), ZFNs (Zinc Finger Nucleases), RNA interference (RNAi), Pime editing, Base editing and double-stranded break-free (DSB-free). These technologies enable precise genetic modifications, offering opportunities to enhance traits that reduce environmental impact and optimize metabolic pathways. Additionally, various nutrition-related measures have shown promise in mitigating methane emissions to varying extents. This review aims to present a future-oriented viewpoint on reducing methane emissions from ruminants by leveraging CRISPR/Cas9 technology to engineer the microbial consortia within the rumen. The ultimate objective is to develop sustainable livestock production methods that effectively decrease methane emissions, while maintaining animal health and productivity.

Using CRISPR-Cas9 to create knockout mutants in insects

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) is a potent genome editing tool that has rapidly revolutionized research in natural science disciplines. The CRISPR/Cas9 system surpasses traditional zinc finger nucleases and transcription activator-like effector nucleases because of its simplicity in design, cost-effectiveness, and labor intensity. The CRISPR/Cas9-mediated genome editing system has been used to generate precise and targeted mutations in many different organisms, including insects. However, there is still a lack of a comprehensive review that systematically summarizes the successful utilization of the CRISPR/Cas9 system in generating knockout (KO) mutations in insects (both model and non-model). Thus, this review provides a comprehensive analysis of the current advancements in CRISPR/Cas9-based KO mutants in insects, including both model and non-model species. We also discussed the limitations of the CRISPR/Cas9 system. Finally, we discuss the biosafety concerns associated with the release of CRISPR/Cas9-edited insects into the environment and draw inroads and insights into the future application of the CRISPR/Cas9 system in insects.

Application of genome editing in plant reproductive biology: recent advances and challenges.

This comprehensive review underscores the application of genome editing in plant reproductive biology, including recent advances and challenges associated with it. Genome editing (GE) is a powerful technology that has the potential to accelerate crop improvement by enabling efficient, precise, and rapid engineering of plant genomes. Over the last decade, this technology has rapidly evolved from the use of meganucleases (homing endonucleases), zinc-finger nucleases, transcription activator-like effector nucleases to the use of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas), which has emerged as a popular GE tool in recent times and has been extensively used in several organisms, including plants. GE has been successfully employed in several crops to improve plant reproductive traits. Improving crop reproductive traits is essential for crop yields and securing the world's food supplies. In this review, we discuss the application of GE in various aspects of plant reproductive biology, including its potential application in haploid induction, apomixis, parthenocarpy, development of male sterile lines, and the regulation of self-incompatibility. We also discuss current challenges and future prospects of this technology for crop improvement, focusing on plant reproduction.

Genome Editing Approaches Using Zinc Finger Nucleases (ZFNs) for the Treatment of Motor Neuron Diseases

Genome Editing Approaches Using Zinc Finger Nucleases (ZFNs) for the Treatment of Motor Neuron Diseases

Genome editing: An insight into disease resistance, production efficiency, and biomedical applications in livestock.

One of the primary concerns for the survival of the human species is the growing demand for food brought on by an increasing global population. New developments in genome-editing technology present promising opportunities for the growth of wholesome and prolific farm animals. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. Genome editing entails modifying genetic material by removing, adding, or manipulating particular DNA sequences from a particular locus in a way that does not happen naturally. The three primary genome editors are CRISPR/Cas 9, TALENs, and ZFNs. Each of these enzymes is capable of precisely severing nuclear DNA at a predetermined location. One of the most effective inventions is base editing, which enables single base conversions without the requirement for a DNA double-strand break (DSB). As reliable methods for precise genome editing in studies involving animals, cytosine and adenine base editing are now well-established. Effective zygote editing with both cytosine and adenine base editors (ABE) has resulted in the production of animal models. Both base editors produced comparable outcomes for the precise editing of point mutations in somatic cells, advancing the field of gene therapy. This review focused on the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of ZFNs, TALENs, and CRISPR/Cas9 base editors, and prime editing in diverse lab and farm animals. Additionally, we address the methodologies that can be used for gene regulation, base editing, and epigenetic alterations, as well as the significance of genome editing in animal models to better reflect real disease. We also look at methods designed to increase the effectiveness and precision of gene editing tools. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. This review is an overview of the existing knowledge of the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of zinc finger nucleases (ZFNs), transcription-activator-like endonucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas 9), base editors and prime editing in diverse lab and farm animals, which will offer better and healthier products for the entire human race.

β-Thalassemia gene editing therapy: Advancements and difficulties.

β-Thalassemia is the world's number 1 single-gene genetic disorder and is characterized by suppressed or impaired production of β-pearl protein chains. This results in intramedullary destruction and premature lysis of red blood cells in peripheral blood. Among them, patients with transfusion-dependent β-thalassemia face the problem of long-term transfusion and iron chelation therapy, which leads to clinical complications and great economic stress. As gene editing technology improves, we are seeing the dawn of a cure for the disease, with its reduction of ineffective erythropoiesis and effective prolongation of survival in critically ill patients. Here, we provide an overview of β-thalassemia distribution and pathophysiology. In addition, we focus on gene therapy and gene editing advances. Nucleic acid endonuclease tools currently available for gene editing fall into 3 categories: zinc finger nucleases, transcription activator-like effector nucleases, and regularly interspaced short palindromic repeats (CRISPR-Cas9) nucleases. This paper reviews the exploratory applications and exploration of emerging therapeutic tools based on 3 classes of nucleic acid endonucleases in the treatment of β-thalassemia diseases.

Gaining momentum: stem cell therapies for HIV cure.

Durable HIV-1 remission has been reported in a person who received allogeneic stem cell transplants (SCTs) involving CCR5 Δ32/Δ32 donor cells. Much of the reduction in HIV-1 burden following allogeneic SCT with or without donor cells inherently resistant to HIV-1 infection is likely due to cytotoxic graft-versus-host effects on residual recipient immune cells. Nonetheless, there has been growing momentum to develop and implement stem cell therapies that lead to durable long-term antiretroviral therapy (ART)-free remission without the need for SCT. Most current research leverages gene editing techniques to modify hematopoietic stem cells which differentiate into immune cells capable of harboring HIV-1. Approaches include targeting genes that encode HIV-1 co-receptors using Zinc Finger Nucleases (ZFN) or CRISPR-Cas-9 to render a pool of adult or progenitor cells resistant to de-novo infection. Other strategies involve harnessing multipotent mesenchymal stromal cells to foster immune environments that can more efficiently recognize and target HIV-1 while promoting tissue homeostasis. Many of these strategies are currently in a state of infancy or adolescence; nonetheless, promising preclinical and first-in-human studies have been performed, providing further rationale to focus resources on stem cell therapies.

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.

Engineering of Zinc Finger Nucleases Through Structural Modeling Improves Genome Editing Efficiency in Cells.

Genome Editing is widely used in biomedical research and medicine. Zinc finger nucleases (ZFNs) are smaller in size than transcription activator-like effector (TALE) nucleases (TALENs) and CRISPR-Cas9. Therefore, ZFN-encoding DNAs can be easily packaged into a viral vector with limited cargo space, such as adeno-associated virus (AAV) vectors, for in vivo and clinical applications. ZFNs have great potential for translational research and clinical use. However, constructing functional ZFNs and improving their genome editing efficiency is extremely difficult. Here, the efficient construction of functional ZFNs and the improvement of their genome editing efficiency using AlphaFold, Coot, and Rosetta are described. Plasmids encoding ZFNs consisting of six fingers using publicly available zinc-finger resources are assembled. Two functional ZFNs from the ten ZFNs tested are successfully obtained. Furthermore, the engineering of ZFNs using AlphaFold, Coot, or Rosetta increases the efficiency of genome editing by 5%, demonstrating the effectiveness of engineering ZFNs based on structural modeling.

Isolation, Characterization and Genome-Wide Identification of Cys-2/His-2 Type Zinc Finger Nuclease, a Transcription Activator in Saccharum Complex Under Abiotic Stress Conditions

Isolation, Characterization and Genome-Wide Identification of Cys-2/His-2 Type Zinc Finger Nuclease, a Transcription Activator in Saccharum Complex Under Abiotic Stress Conditions

Biotechnological Innovations in Sustainable Food Production

This review article explores the critical role of biotechnology in addressing the global food security crisis aggravated by the COVID-19 pandemic and Eastern European conflict. It starts by examining the profound impacts of these events on food supply chains and pricing, supported by relevant statistical evidence. The study then discusses agricultural biotechnology, comparing conventional and sustainable agriculture, with an emphasis on genome editing and modification techniques such as Zinc Finger Nucleases (ZFNs), Oligonucleotide-directed mutagenesis (ODM), and CRISPR. The application of RNA interference in agriculture and microbial biotechnology in promoting soil health and sustainable food production is discussed. Furthermore, the review shifts focus to the utilization of microbial biotechnology for soil health enhancement and sustainable food production, encompassing the development of pest-resistant and drought-tolerant crops and biotechnological methods for optimizing water use efficiency. Additionally, it examines biotechnology's applications in food processing, including enhancing nutritional content and improving shelf life and safety. The European Green Deal is analyzed, particularly its influence on agriculture through strategies like Farm to Fork, Biodiversity, and Circular Economy. Finally, the review concludes by addressing policy considerations, ethical challenges, and the necessity of international cooperation in biotechnology research, exemplified by projects like the Golden Rice and Heat-Tolerant Maize for Asia (HTMA), highlighting the multidisciplinary nature and global significance of biotechnological innovations in ensuring sustainable food production.

Gene Editing's Sharp Edge: Understanding Zinc Finger Nucleases (ZFN), Transcription Activator-Like Effector Nucleases (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

At present, the main gene editing tools encompass TREN, Zinc Finger Nucleases (ZFN), clustered regularly interspaced short palindromic repeats (CRISPR), and Transcription Activator-Like Effector Nucleases (TALEN). In this study, we introduce an overview of the three gene editing methodologies and discuss their current clinical applications. In addition, we suggest some trends and future applications within the field of gene editing. ZFNs represent one of the pioneering technologies, demonstrating significant efficacy in mitigating a multitude of genetic diseases and finding applications in agriculture. Yet, this technology contains intricate processes and produces substantial costs when implemented. TALENs have already been employed across various domains. In the medical field, they have been successfully applied in the treatment of leukemia in infants. However, TALENs are being replaced by CRISPR due to the superior efficiency of CRISPR. CRISPR, consisting of six components, exhibits considerable promise in the medical realm, particularly in the context of treating diseases such as Alzheimer's disease (AD). In the realm of genetic engineering, it can collaborate with B cells to rectify specific genes within the human genome, which have been tested in experiments. In the future, it can be used in many fields, including agriculture and nucleic acid testing.