Transcription Activator-like Nucleases Research Articles

A strategy for improving the mechanical properties of silk fibers through the combination of genetic manipulation and zinc ion crosslinking

Silk fiber is generally considered an excellent biological material due to its good biocompatibility, morphological plasticity and biodegradability. Previously, the construction of silkworm silk gland bioreactors based on the piggyBac transposon has been optimized. However, the inserted exogenous genes have problems such as position uncertainty, and expression is not strictly controlled. Here, we applied transcription activator-like effector nuclease (TALEN)-mediated homology-directed repair (HDR) to precisely insert histidine-rich cuticular protein (CP) into silkworm Sericin1 (Ser1) gene. The Ser1-CP fusion protein was successfully secreted into cocoon shell. Subsequently, based on the metal coordination ability of the histidine imidazole group, we crosslinked cocoon with metal ions in vitro. In this strategy, the mechanical properties of the fused silk fibers with crosslinked Zn2+ improved, and the maximum breaking stress of the crosslinked Zn2+-fused silk fibers was 23.5 % greater than that of the wild-type fibers. Analysis of the secondary structure of the silk protein showed that the fused silk fibers crosslinked with Zn2+ had more β-sheet structures. This study pioneered a method of improving the mechanical properties of silk fibers by crosslinking metal ions with fused exogenous proteins and expanded the application value of silk gland bioreactors in the development of novel biomaterials.

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.

Disruption of <i>CYP88B1</i> by transcription activator-like effector nuclease in potato and potential use to produce useful saponins

Disruption of <i>CYP88B1</i> by transcription activator-like effector nuclease in potato and potential use to produce useful saponins

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.

Genome editing in angiosperm chloroplasts: targeted DNA double-strand break and base editing.

Chloroplasts are organelles that are derived from a photosynthetic bacterium and have their own genome. Genome editing is a recently developing technology that allows for specific modifications of target sequences. The first successful application of genome editing in chloroplasts was reported in 2021, and since then, this research field has been expanding. Although the chloroplast genome of several dicot species can be stably modified by a conventional method, which involves inserting foreign DNAs into the chloroplast genome via homologous recombination, genome editing offers several advantages over this method. In this review, we introduce genome editing methods targeting the chloroplast genome and describe their advantages and limitations. So far, CRISPR/Cas systems are inapplicable for editing the chloroplast genome because guide RNAs, unlike proteins, cannot be efficiently delivered into chloroplasts. Therefore, protein-based enzymes are used to edit the chloroplast genome. These enzymes contain a chloroplast-transit peptide, the DNA-binding domain of transcription activator-like effector nuclease (TALEN), or a catalytic domain that induces DNA modifications. To date, genome editing methods can cause DNA double-strand break or introduce C:G-to-T:A and A:T-to-G:C base edits at or near the target sequence. These methods are expected to contribute to basic research on the chloroplast genome in many species and to be fundamental methods of plant breeding utilizing the chloroplast genome.

Production of a heterozygous exon skipping model of common marmosets using gene-editing technology

Nonhuman primates (NHPs), which are closely related to humans, are useful in biomedical research, and an increasing number of NHP disease models have been reported using gene editing. However, many disease-related genes cause perinatal death when manipulated homozygously by gene editing. In addition, NHP resources, which are limited, should be efficiently used. Here, to address these issues, we developed a method of introducing heterozygous genetic modifications into common marmosets by combining Platinum transcription activator-like effector nuclease (TALEN) and a gene-editing strategy in oocytes. We succeeded in introducing the heterozygous exon 9 deletion mutation in the presenilin 1 gene, which causes familial Alzheimer’s disease in humans, using this technology. As a result, we obtained animals with the expected genotypes and confirmed several Alzheimer’s disease-related biochemical changes. This study suggests that highly efficient heterozygosity-oriented gene editing is possible using TALEN and oocytes and is an effective method for producing genetically modified animals.

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.

MAVS disruption impairs downstream signaling and results in higher virus replication levels of salmonid alphavirus subtype 3 but not infectious pancreatic necrosis virus in vitro.

The mitochondrial anti-viral signaling (MAVS) protein is an intermediary adaptor protein of retinoic acid-inducible gene-1 (RIG-I) like receptor (RLR) signaling, which activates the transcription factor interferon (IFN) regulatory factor 3 (IRF3) and NF-kB to produce type I IFNs. MAVS expression has been reported in different fish species, but few studies have shown its functional role in anti-viral responses to fish viruses. In this study, we used the transcription activator-like effector nuclease (TALEN) as a gene editing tool to disrupt the function of MAVS in Chinook salmon (Oncorhynchus tshawytscha) embryonic cells (CHSE) to understand its role in induction of interferon I responses to infections with the (+) RNA virus salmonid alphavirus subtype 3 (SAV-3), and the dsRNA virus infectious pancreatic necrosis virus (IPNV) infection. A MAVS-disrupted CHSE clone with a 7-aa polypeptide (GVFVSRV) deletion mutation at the N-terminal of the CARD domain infected with SAV-3 resulted in significantly lower expression of IRF3, IFNa, and ISGs and increased viral titer (1.5 log10) compared to wild-type. In contrast, the IPNV titer in MAVS-disrupted cells was not different from the wild-type. Furthermore, overexpression of salmon MAVS in MAVS-disrupted CHSE cells rescued the impaired type I IFN-mediated anti-viral effect against SAV-3.

Latest advances on new promising molecular-based therapeutic approaches for Huntington's disease.

Huntington's disease (HD) is a devastating, autosomal-dominant inherited, neurodegenerative disorder characterized by progressive motor deficits, cognitive impairments, and neuropsychiatric symptoms. It is caused by excessive cytosine-adenine-guanine (CAG) trinucleotide repeats within the huntingtin gene (HTT). Presently, therapeutic interventions capable of altering the trajectory of HD are lacking, while medications for abnormal movement and psychiatric symptoms are limited. Numerous pre-clinical and clinical studies have been conducted and are currently underway to test the efficacy of therapeutic approaches targeting some of these mechanisms with varying degrees of success. In this review, we update the latest advances on new promising molecular-based therapeutic strategies for this disorder, including DNA-targeting techniques such as zinc-finger proteins, transcription activator-like effector nucleases, and CRISPR/Cas9; post-transcriptional huntingtin-lowering approaches such as RNAi, antisense oligonucleotides, and small-molecule splicing modulators; and novel methods to clear the mHTT protein, such as proteolysis-targeting chimeras. We mainly focus on the ongoing clinical trials and the latest pre-clinical studies to explore the progress of emerging potential HD therapeutics.

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.

IGF-1 Genome-Edited Human MSCs Exhibit Robust Anti-Arthritogenicity in Collagen-Induced Arthritis.

Stem cell therapy stands out as a promising avenue for addressing arthritis treatment. However, its therapeutic efficacy requires further enhancement. In this study, we investigated the anti-arthritogenic potential of human amniotic mesenchymal stem cells (AMM) overexpressing insulin-like growth factor 1 (IGF-1) in a collagen-induced mouse model. The IGF-1 gene was introduced into the genome of AMM through transcription activator-like effector nucleases (TALENs). We assessed the in vitro immunomodulatory properties and in vivo anti-arthritogenic effects of IGF-1-overexpressing AMM (AMM/I). Co-culture of AMM/I with interleukin (IL)-1β-treated synovial fibroblasts significantly suppressed NF-kB levels. Transplantation of AMM/I into mice with collagen-induced arthritis (CIA) led to significant attenuation of CIA progression. Furthermore, AMM/I administration resulted in the expansion of regulatory T-cell populations and suppression of T-helper-17 cell activation in CIA mice. In addition, AMM/I transplantation led to an increase in proteoglycan expression within cartilage and reduced infiltration by inflammatory cells and also levels of pro-inflammatory factors including cyclooxygenase-2 (COX-2), IL-1β, NF-kB, and tumor necrosis factor (TNF)-α. In conclusion, our findings suggest that IGF-1 gene-edited human AMM represent a novel alternative therapeutic strategy for the treatment of arthritis.

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.

Variant in EZR leads to defects in lens development

ABSTRACT Background Congenital cataract is a common cause of blindness. Genetic factors always play important role. Material and Methods This study identified a novel missense variant (c.1412C>T (p.P471L)) in the EZR gene in a four-generation Chinese family with nuclear cataract by linkage analysis and whole-exome sequencing. A knockout study in zebrafish using transcription activator-like effector nucleases was carried out to gain insight into candidate gene function. Results Conservative and functional prediction suggests that the P-to-L substitution may impair the function of the human ezrin protein. Histology showed developmental delays in the ezrin-mutated zebrafish, manifesting as multilayered lens epithelial cells. Immunohistochemistry revealed abnormal proliferation patterns in mutant fish. Conclusions The study suggests that ezrin may be involved in the enucleation and differentiation of lens epithelial cells.

The pathogenic mechanism of syndactyly type V identified in a Hoxd13Q50R knock-in mice

Syndactyly type V (SDTY5) is an autosomal dominant extremity malformation characterized by fusion of the fourth and fifth metacarpals. In the previous publication, we first identified a heterozygous missense mutation Q50R in homeobox domain (HD) of HOXD13 in a large Chinese family with SDTY5. In order to substantiate the pathogenicity of the variant and elucidate the underlying pathogenic mechanism causing limb malformation, transcription-activator-like effector nucleases (TALEN) was employed to generate a Hoxd13Q50R mutant mouse. The mutant mice exhibited obvious limb malformations including slight brachydactyly and partial syndactyly between digits 2–4 in the heterozygotes, and severe syndactyly, brachydactyly and polydactyly in homozygotes. Focusing on BMP2 and SHH/GREM1/AER-FGF epithelial mesenchymal (e-m) feedback, a crucial signal pathway for limb development, we found the ectopically expressed Shh, Grem1 and Fgf8 and down-regulated Bmp2 in the embryonic limb bud at E10.5 to E12.5. A transcriptome sequencing analysis was conducted on limb buds (LBs) at E11.5, revealing 31 genes that exhibited notable disparities in mRNA level between the Hoxd13Q50R homozygotes and the wild-type. These genes are known to be involved in various processes such as limb development, cell proliferation, migration, and apoptosis. Our findings indicate that the ectopic expression of Shh and Fgf8, in conjunction with the down-regulation of Bmp2, results in a failure of patterning along both the anterior-posterior and proximal-distal axes, as well as a decrease in interdigital programmed cell death (PCD). This cascade ultimately leads to the development of syndactyly and brachydactyly in heterozygous mice, and severe limb malformations in homozygous mice. These findings suggest that abnormal expression of SHH, FGF8, and BMP2 induced by HOXD13Q50R may be responsible for the manifestation of human SDTY5.

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.

Role of CRISPR in Crop Improvement: A Review

The meals and agriculture sectors have witnessed good sized advancements due to the state-of-the-art improvements in agricultural biotechnology and genetic engineering, that have more advantageous the essential characteristics of plant agronomic tendencies. A extensively used technique for inducing focused deletions, insertions, and precise sequence adjustments throughout numerous species and mobile kinds is collection-particular nucleases (SSNs)-based centered genome editing. Commercial adoption of genome modifying gear, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and siRNA-mediated RNA interference, has been significant. However, the whole landscape of lifestyles sciences underwent a paradigm shift with the invention of the CRISPR/Cas9 machine as a flexible tool for genome modifying. Initially recognized as a virological defense DNA segment in bacteria and archaea, the clustered Regularly interspaced short palindromic repeats (CRISPR) machine has revolutionized molecular biology. Through modern molecular organic strategies, CRISPR/Cas9 enables unique modifications in any crop species. Its efficacy, reproducibility, and specificity have earned CRISPR/Cas9 the moniker of a "leap forward" in biotechnology.

Custom-designed, mass silk production in genetically engineered silkworms.

Genetically engineered silkworms have been widely used to obtain silk with modified characteristics especially by introducing spider silk genes. However, these attempts are still challenging due to limitations in transformation strategies and difficulties in integration of the large DNA fragments. Here, we describe three different transformation strategies in genetically engineered silkworms, including transcription-activator-like effector nuclease (TALEN)-mediated fibroin light chain (FibL) fusion (BmFibL-F), TALEN-mediated FibH replacement (BmFibH-R), and transposon-mediated genetic transformation with the silk gland-specific fibroin heavy chain (FibH) promoter (BmFibH-T). As the result, the yields of exogenous silk proteins, a 160 kDa major ampullate spidroin 2 (MaSp2) from the orb-weaving spider Nephila clavipes and a 226 kDa fibroin heavy chain protein (EvFibH) from the bagworm Eumeta variegate, reach 51.02 and 64.13% in BmFibH-R transformed cocoon shells, respectively. Moreover, the presence of MaSp2 or EvFibH significantly enhances the toughness of genetically engineered silk fibers by ∼86% in BmFibH-T and ∼80% in BmFibH-R silkworms, respectively. Structural analysis reveals a substantial ∼40% increase in fiber crystallinity, primarily attributed to the presence of unique polyalanines in the repetitive sequences of MaSp2 or EvFibH. In addition, RNA-seq analysis reveals that BmFibH-R system only causes minor impact on the expression of endogenous genes. Our study thus provides insights into developing custom-designed silk production using the genetically engineered silkworm as the bioreactor.