Transcription Activator-like Nucleases Research Articles

Genome Editing in Mouse and Rat by Electroporation.

Many genome-edited mouse and rat strains have been produced using engineered endonucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Especially, CRISPR-Cas9 is powerful tool that can be easy, rapid, and high-efficiency-produced new genome-edited strains. Furthermore, new technique, Technique for Animal Knockout system by Electroporation (TAKE), efficiently accelerate production of new strains by direct nuclease introduction into intact embryos using electroporation. This chapter presents a latest technical information in the production of genome-edited mouse and rat by TAKE method.

Construction and Evaluation of Zinc Finger Nucleases.

Zinc finger nucleases (ZFNs) are programmable nucleases that have contributed significantly to past genome-editing research. They are now utilized much less owing to the advent of transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein system (CRISPR-Cas). These new methods allow for easier generation of reagents that target genomic sequences of interest and are less labor-intensive than ZFNs at targeting desired sequences. However, fundamental ZFN patents have expired, enabling a wide range of their distribution for clinical and industrial applications. This article introduces a ZFN construction protocol that uses bacterial one-hybrid (B1H) selection and single-strand annealing (SSA) assay.

Determining On‐Target, Off‐Target, and Copy Number Status of Transgenic Events After CRISPR/Cas9 Targeted AAVS1 Safe‐Harbor Modification of iPSCs Using Double‐Control Quantitative Copy Number PCR

Double-control quantitative copy number PCR (dc-qcnPCR) is a recently described tool that can be used to quantify donor DNA insertions in genetically modified monoclonal cell lines. In conjunction with an insert-confirmation PCR, the technique can quickly and easily identify clones containing on-target heterozygous or homozygous donor DNA integrations and exclude off-target insertions. The genetic manipulation of immortal cell lines is a versatile tool to elucidate cellular signaling pathways and protein functions. Despite recent advances in the precision of genetic engineering tools such as CRISPR/Cas9, transcription-activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs), it is still essential to verify the accurate insertion of the sequence of interest (donor DNA) into the targeted genomic DNA (gDNA) locus. This precise integration into a genetic safe harbor, and exclusion of the donor DNA from functionally relevant genes, can ensure normal cellular functionality. Current methods to analyze the specificity of donor DNA insertions either are cost-prohibitive or create dependency on manufacturers for assay design and production. The dc-qcnPCR method is a simple, yet powerful, approach that can be prepared and carried out in any laboratory equipped with standard molecular biology supplies. Here we provide step-by-step instructions to prepare and perform the dc-qcnPCR, and its companion insert-confirmation PCR, to determine donor DNA insertion numbers in monoclonal cell lines genetically modified through CRISPR/Cas9. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genetic modification at AAVS1 safe harbor in induced pluripotent stem cells (IMR90-4) using CRISPR/Cas9: from plasmid design to monoclonal expansion Support Protocol 1: Measurement of Gaussia luciferase activity to verify reporter protein functionality Support Protocol 2: Verification of monoclonal expansion using immunofluorescence. Basic Protocol 2: Insert-confirmation PCR Basic Protocol 3: Design and preparation of double-control quantitative copy number PCR reagents and quantification of donor DNA integrations in genetically modified monoclonal cells.

A Method for Precisely Identifying Modifications to Plant Mitochondrial Genomes by mitoTALENs.

The ability to transform plant mitochondrial genomes has many benefits. Although delivery of foreign DNA to mitochondria is presently very difficult, it is now possible to knock out mitochondrial genes using mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs). Such knockouts have been achieved by a genetic transformation of mitoTALENs encoding genes into the nuclear genome. Previous studies have shown that double-strand breaks (DSBs) induced by mitoTALENs are repaired by ectopic homologous recombination. As a result of DNA repair by homologous recombination, a portion of the genome containing the mitoTALEN target site is deleted. The deletion and repair process cause the mitochondrial genome to become more complex. Here, we describe a method for identifying the ectopic homologous recombination events that occur following the repair of double-strand breaks induced by mitoTALENs.

Protein Arginine Methyltransferase 5 Is Necessary for Embryonic Development in Medaka Oryzias latipes

Protein arginine methyltransferase 5 (Prmt5), conserved from yeast to humans, catalyzes arginine’s dimethylation in proteins. Prmt5 is necessary for embryonic development in mice because it maintains embryonic stem cells. However, the embryos of zebrafish (Danio rerio) remain viable with a deficiency in germ cells and sexual development after the knockout of prmt5. Therefore, it was considered whether prmt5 is dispensable during embryogenesis in fish. Medaka (Oryzias latipes), another model fish organism, was used in this experiment. The medaka prmt5 was mutated with Transcription Activator-Like Effector Nucleases (TALEN) causing the premature stopping of transcription. None of the homozygous prmt5 mutant fish were viable, only the heterozygous offspring survived. Quantitative reverse transcription-polymerase chain reaction (qPCR) results showed a significant decrease in octamer-binding transcription factor 4 (oct4), homeobox transcription factor nanog (nanog), vasa, B-cell CLL/lymphoma 2 (bcl2), and the ratio of bcl2 to bax (bcl2 associated x), and a significant increase in caspase3 and caspase8 in the embryos of the heterozygous prmt5 mutant compared with that of the wild type. The results showed that the mutation of prmt5 caused down-regulation of the genes functioning in stemness and up-regulation of the genes in the cascade of cell death. These results suggested that prmt5 is necessary for embryogenesis via maintaining stemness and repressing apoptosis in medaka.

Detection of chromosomal alteration after infusion of gene-edited allogeneic CAR T cells

A chromosome 14 inversion was found in a patient who developed bone marrow aplasia following treatment with allogeneic chimeric antigen receptor (CAR) Tcells containing gene edits made with transcription activator-like effector nucleases (TALEN). TALEN editing sites were not involved at either breakpoint. Recombination signal sequences (RSSs) were found suggesting recombination-activating gene (RAG)-mediated activity. The inversion represented a dominant clone detected in the context of decreasing absolute CAR Tcell and overall lymphocyte counts. The inversion was not associated with clinical consequences and wasnot detected in the drug product administered to this patient or in any drug product used in this or other trials using the same manufacturing processes. Neither was the inversion detected in this patient at earlier time points or in any other patient enrolled in this or other trials treated with this or other product lots. This case illustrates that spontaneous, possibly RAG-mediated, recombination events unrelated to gene editing can occur in adoptive cell therapy studies, emphasizes the need for ruling out off-target gene editing sites, and illustrates that other processes, such as spontaneous V(D)J recombination, can lead to chromosomal alterations in infused cells independent of gene editing.

Challenges, Recent Advances and Perspectives in the Treatment of Human Cytomegalovirus Infections.

Human cytomegalovirus (HCMV) is ubiquitous worldwide and elicits global health problems. The diseases associated with HCMV are a serious threat to humans, especially for the sick, infant, elderly and immunocompromised/immunodeficient individuals. Although traditional antiviral drugs (e.g., ganciclovir, valganciclovir, cidofovir, foscarnet) can be used to treat or prevent acute HCMV infections, their efficacy is limited because of toxicity, resistance issues, side effects and other problems. Fortunately, novel drugs (e.g., letermovir and maribavir) with less toxicity and drug/cross-resistance have been approved and put on the market in recent years. The nucleic acid-based gene-targeting approaches including the external guide sequences (EGSs)-RNase, the clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPRs-associated protein 9 (Cas9) system and transcription activator-like effector nucleases (TALENs) have been investigated to remove both lytic and latent CMV in vitro and/or in vivo. Cell therapy including the adoptive T cell therapy (ACT) and immunotherapy have been tried against drug-resistant and recurrent HCMV in patients receiving hematopoietic stem cell transplantation (HSCT) or solid organ transplant (SOT), and they have also been used to treat glioblastoma (GBM) associated with HCMV infections. These newly developed antiviral strategies are expected to yield fruitful results and make a significant contribution to the treatment of HCMV infections. Despite this progress, the nucleic acid-based gene-targeting approaches are still under study for basic research, and cell therapy is adopted in a small study population size or only successful in case reports. Additionally, no current drugs have been approved to be indicated for latent infections. Therefore, the next strategy is to develop antiviral strategies to elevate efficacy against acute and/or latent infections and overcome challenges such as toxicity, resistance issues, and side effects. In this review, we would explore the challenges, recent advances and perspectives in the treatment of HCMV infections. Furthermore, the suitable therapeutic strategies as well as the possibility for compassionate use would be evaluated.

CRISPR/Cas9 Mediated Therapeutic Approach in Huntington's Disease.

The pathogenic mechanisms of these diseases must be well understood for the treatment of neurological disorders such as Huntington's disease. Huntington's Disease (HD), a dominant and neurodegenerative disease, is characterized by the CAG re-expansion that occurs in the gene encoding the polyglutamine-expanded mutant Huntingtin (mHTT) protein. Genome editing approaches include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats/Caspase 9 (CRISPR/Cas9) systems. CRISPR/Cas9 technology allows effective gene editing in different cell types and organisms. Through these systems are created isogenic control of human origin induced pluripotent stem cells (iPSCs). In human and mouse models, HD-iPSC lines can be continuously corrected using these systems. HD-iPSCs can be corrected through the CRISPR/Cas9 system and the cut-and-paste mechanism using isogenic control iPSCs. This mechanism is a piggyBac transposon-based selection system that can effectively switch between vectors and chromosomes. In studies conducted, it has been determined that in neural cells derived from HD-iPSC, there are isogenic controls as corrected lines recovered from phenotypic abnormalities and gene expression changes. It has been determined that trinucleotide repeat disorders occurring in HD can be cured by single-guide RNA (sgRNA) and normal exogenous DNA restoration, known as the single guideline RNA specific to Cas9. The purpose of this review in addition to give general information about HD, a neurodegenerative disorder is to explained the role of CRISPR/Cas9 system with iPSCs in HD treatment.

Development of a Transcriptional Activator-Like Effector Protein to Accurately Discriminate Single Nucleotide Difference.

Transcriptional activator-like effector (TALE), a DNA-binding protein, is widely used in genome editing. However, the recognition of the target sequence by the TALE is adversely affected by the number of mismatches. Therefore, the association constant of DNA-TALE complex formation can be controlled by appropriately introducing a mismatch into the TALE recognition sequence. This study aimed to construct a TALE that can distinguish a single nucleotide difference. Our results show that a single mismatch present in repeats 2 or 3 of TALE did not interfere with the complex formation with DNA, whereas continuous mismatches present in repeats 2 and 3 significantly reduced association with the target DNA. Based on these findings, we constructed a detection system of the one nucleotide difference in gene with high accuracy and constructed a TALE-nuclease (TALEN) that selectively cleaves DNA with a single mismatch.

Dual CXCR4/IL-10 Gene-Edited Human Amniotic Mesenchymal Stem Cells Exhibit Robust Therapeutic Properties in Chronic Wound Healing

Although stem cells have attracted attention as a novel therapeutic solution for tissue regeneration, their minimal efficacy remains controversial. In the present study, we aimed to investigate the enhanced therapeutic property of CXCR4/IL-10 dual angiogenic/anti-inflammatory gene knock-in amniotic mesenchymal stem cells (AMM) in a wound-healing model. Dual CXCR4 and IL-10 genes were inserted into the AMM genome using transcription-activator-like effector nuclease (TALEN). Matrigel tube formation and anti-inflammatory effects were assessed in vitro, and efficacy was tested in vivo in a diabetic wound-healing model. CXCR4/IL-10-expressing amniotic MSCs (AMM/CI) strongly expressed CXCR4 and IL-10 genes and robustly promoted tube formation and anti-inflammatory potential. AMM/CI transplantation resulted in accelerated wound healing, as well as high engraftment and re-epithelialization potential. Transplanted AMM/CI also exhibited high angiogenic and decreased pro-inflammatory gene expression in the wound tissue, indicating direct therapeutic effects on wound healing. Taken together, these data indicate that dual angiogenic/anti-inflammatory gene knock-in may be a novel approach to enhance the therapeutic effects of stem cells, and transplantation of AMM/CI can be an alternative therapeutic option in chronic wound healing.

Genome Editing and Protein Energy Malnutrition.

Protein-energy malnutrition is a state of disordered catabolism resulting from metabolic derangements or starvation. It is associated with chronic disease, hypoglycemia, hypothermia, serious infections, and even an increased prevalence of morbidity and mortality in countries with poor socioeconomic or environmental factors. Adequate food administration is essential to satisfy the main caloric and nutritional demands of humans. The most significant factors seen in the development of protein-energy malnutrition in areas of high incidence, such as underdeveloped countries, are inadequate food and nutrient supplies. It has been well established that one of the strategies to alleviate undernourishment is the biofortification of staple crops. This is because vegetables and plants are significant sources of crucial nutrients for human growth and development. To enhance plant nutrition, recent tactics aim to formulated balanced and diverse diets with acceptable levels ofvitamins and minerals that benefit human health. New advances in plant biotechnology and animal productivity could control key enzymes in several metabolic pathways, enriching important nutrients such as iron and vitamins and decreasing the content of disadvantageous compounds such as acrylamide-forming amino acids and phytic acids. Numerous biofortified crops such as rice, maize, and wheat have been created to resolve the problem of nutrition deficiencies. Some examples of these methodologies are genome editing engineered nucleases, transcriptional activator-like effector nucleases, zinc finger nucleases, and clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease which have been created and widely studied for their application, efficiency, and specificity.

Genome Editing and Pathological Cardiac Hypertrophy.

Three major genome editing tools, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeat (CRISPR) systems, are increasingly important technologies used in the study and treatment of hereditary myocardial diseases. Germ cell genome editing and modification can permanently eliminate monogenic cardiovascular disease from the offspring of affected families and the next generation, although ethically controversial. Somatic genome editing may be a promising method for the treatment of hereditary cardiomyopathy various diseases for which gene knockout is favorable and can also treat people who are already ill, although there are currently some technical challenges. This chapter describes the application of genome editing in the experimental studies and treatment of hypertrophic cardiomyopathy as well as other cardiomyopathies.

Plant genome modification: from induced mutagenesis to genome editing.

The snowballing growth of scientif ic data obtained using modern techniques of genome editing (GE) calls for their critical evaluation and comparison against previously applied methods such as induced mutagenesis, which was a leading method of genome modif ication for many decades of the past century, and its application has resulted in a huge diversity of cultivars. However, this method was relatively long and included a number of stages from inducing multiple mutations using different mutagenic factors to crossing and selecting the most valuable cultivars for several generations. A new technology of genetic engineering and transgenesis enabled us to radically reduce the time required to obtain a new genetically-modif ied cultivar to one generation and make the modif ication process more effective and targeted. The main drawback of this approach was that an introduced transgene might uncontrollably affect the other genes of a recipient plant, which led to the limitations imposed on transgenesis application in many countries. These limitations have been effectively surmounted thanks to the development of GE techniques allowing for a precise modif ication within a single gene that in many characteristics make it similar to a natural allele (especially when it comes to ribonucleoprotein complexes), which has paved the way for wide application of GE in routine breeding. The paper reviews the main stages of GE development in its application in plants. It provides short descriptions of different GE techniques, including those using protein editors such as zinc-f inger and transcription activator-like effector nucleases (TALEN), and the CRISPR/Cas9 technology. It lists a number of achievements in using GE to produce new cultivars of higher yield that are resistant to unfavorable factors and have good nutritional properties. The review also considers the de novo domestication approach, which allows for faster obtaining of new cultivars from natural varieties. In the conclusion, the future ways of GE development are discussed.

In Vivo Hematopoietic Stem Cell Genome Editing: Perspectives and Limitations

The tremendous evolution of genome-editing tools in the last two decades has provided innovative and effective approaches for gene therapy of congenital and acquired diseases. Zinc-finger nucleases (ZFNs), transcription activator- like effector nucleases (TALENs) and CRISPR-Cas9 have been already applied by ex vivo hematopoietic stem cell (HSC) gene therapy in genetic diseases (i.e., Hemoglobinopathies, Fanconi anemia and hereditary Immunodeficiencies) as well as infectious diseases (i.e., HIV), and the recent development of CRISPR-Cas9-based systems using base and prime editors as well as epigenome editors has provided safer tools for gene therapy. The ex vivo approach for gene addition or editing of HSCs, however, is complex, invasive, technically challenging, costly and not free of toxicity. In vivo gene addition or editing promise to transform gene therapy from a highly sophisticated strategy to a "user-friendly' approach to eventually become a broadly available, highly accessible and potentially affordable treatment modality. In the present review article, based on the lessons gained by more than 3 decades of ex vivo HSC gene therapy, we discuss the concept, the tools, the progress made and the challenges to clinical translation of in vivo HSC gene editing.

Genome editing in plants

Genome editing technologies have brought dramatic changes in many fields of research, including plant sciences. Zinc finger nuclease, transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) are key players in genome editing and have been developed for targeted mutagenesis. To apply genome editing to plants, optimization and development of several technologies to overcome plant-specific hurdles has been required. In this review, we highlight recent topics in the plant genome editing field in Japan, ranging from the development of a new genome editing tool to commercial applications of genome edited plants. Such achievements contribute greatly to the development of plant genome research and its application to plant breeding.

CRISPR-Cas9 Technology for the Creation of Biological Avatars Capable of Modeling and Treating Pathologies: From Discovery to the Latest Improvements.

This is a spectacular moment for genetics to evolve in genome editing, which encompasses the precise alteration of the cellular DNA sequences within various species. One of the most fascinating genome-editing technologies currently available is Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated protein 9 (CRISPR-Cas9), which have integrated deeply into the research field within a short period due to its effectiveness. It became a standard tool utilized in a broad spectrum of biological and therapeutic applications. Furthermore, reliable disease models are required to improve the quality of healthcare. CRISPR-Cas9 has the potential to diversify our knowledge in genetics by generating cellular models, which can mimic various human diseases to better understand the disease consequences and develop new treatments. Precision in genome editing offered by CRISPR-Cas9 is now paving the way for gene therapy to expand in clinical trials to treat several genetic diseases in a wide range of species. This review article will discuss genome-editing tools: CRISPR-Cas9, Zinc Finger Nucleases (ZFNs), and Transcription Activator-Like Effector Nucleases (TALENs). It will also encompass the importance of CRISPR-Cas9 technology in generating cellular disease models for novel therapeutics, its applications in gene therapy, and challenges with novel strategies to enhance its specificity.

Spontaneous, Artificial, and Genome Editing-Mediated Mutations in Prunus.

Mutation is a source of genetic diversity widely used in breeding programs for the acquisition of agronomically interesting characters in commercial varieties of the Prunus species, as well as in the rest of crop species. Mutation can occur in nature at a very low frequency or can be induced artificially. Spontaneous or bud sport mutations in somatic cells can be vegetatively propagated to get an individual with the mutant phenotype. Unlike animals, plants have unlimited growth and totipotent cells that let somatic mutations to be transmitted to the progeny. On the other hand, in vitro tissue culture makes it possible to induce mutation in plant material and perform large screenings for mutant's selection and cleaning of chimeras. Finally, targeted mutagenesis has been boosted by the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 and Transcription activator-like effector nuclease (TALEN) editing technologies. Over the last few decades, environmental stressors such as global warming have been threatening the supply of global demand for food based on population growth in the near future. For this purpose, the release of new varieties adapted to such changes is a requisite, and selected or generated Prunus mutants by properly regulated mechanisms could be helpful to this task. In this work, we reviewed the most relevant mutations for breeding traits in Prunus species such as flowering time, self-compatibility, fruit quality, and disease tolerance, including new molecular perspectives in the present postgenomic era including CRISPR/Cas9 and TALEN editing technologies.

Editing the genome of common cereals (Rice and Wheat): techniques, applications, and industrial aspects.

Gene editing techniques have made a significant contribution to the development of better crops. Gene editing enables precise changes in the genome of crops, which can introduce new possibilities for altering the crops' traits. Since the last three decades, various gene editing techniques such as meganucleases, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspersed short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins) have been discovered. In this review, we discuss various gene editing techniques and their applications to common cereals. Further, we elucidate the future of gene-edited crops, their regulatory features, and industrial aspects globally. To achieve this, we perform a comprehensive literature survey using databases such as PubMed, Web of Science, SCOPUS, Google Scholar etc. For the literature search, we used keywords such as gene editing, crop genome modification, CRISPR/Cas, ZFN, TALEN, meganucleases etc. With the advent of the CRISPR/Cas technology in the last decade, the future of gene editing has transitioned into a new dimension. The functionality of CRISPR/Cas in both DNA and RNA has increased through the use of various Cas enzymes and their orthologs. Constant research efforts in this direction have improved the gene editing process for crops by minimizing its off-target effects. Scientists also use computational tools, which help them to design experiments and analyze the results of gene editing experiments in advance. Gene editing has diverse potential applications. In the future, gene editing will open new avenues for solving more agricultural issues and boosting crop production, which may have great industrial prospects.

A Transcriptomic Analysis of Tobacco Leaf with the Functional Loss of the Plastid rpoB Operon Caused by TALEN-Mediated Double-Strand Breakage.

At least two sets of RNA polymerase (RNAP), nucleus (NEP)- and plastid (PEP)-encoded polymerases, recognizing distinct promoters exist in the plastids of land plants. Most plastid genes are regulated by multiple promoters with different strengths in their response to developmental stages and environmental cues. Recently, we applied chloroplast-targeted transcription activator-like effector nuclease (cpTALEN) technology to site-specifically cause double-strand DNA breaks in the rpoB gene of tobacco, which encodes the β-subunit of PEP. The repair of damaged chloroplast DNA (cpDNA) through microhomology-mediated recombination caused the functional loss of the rpoB operon and resulted in the heterotrophic growth of an albino plant. We conducted a genome-wide analysis of the steady state of gene expression in the leaf tissue of PEP-deficient tobacco by RNA-Seq and compared it with that of wild-type plants. The expression of NEP genes was up-regulated in PEP-deficient tobacco; in particular, the level of RpoT3 transcripts encoding the specifically plastid-targeted NEP was significantly increased. Alongside most housekeeping genes, NEP also plays an important role in the regulation of gene expression involved in photosynthesis. In contrast, alongside the photosynthesis-related genes, PEP also plays an important role in the regulation of gene expression involved in some housekeeping functions. Furthermore, the mitochondrial DNA copy number and the level of most mitochondrial protein-coding transcripts were slightly increased in PEP-deficient tobacco. The disruption of PEP function not only affected plastid gene expression, but also nuclear and mitochondrial gene expression. This study demonstrated the intercompartmental retrograde signaling in the regulation of gene expression.

Genome editing technologies, mechanismsand improved production of therapeutic phytochemicals: Opportunities and prospects.

Plants produce a large number of secondary metabolites, known as phytometabolites that may be employed as medicines, dyes, poisons, and insecticides in the field of medicine, agriculture, and industrial use, respectively. The rise of genome management approaches has promised a factual revolution in genetic engineering. Targeted genome editing in living entities permits the understanding of the biological systems very clearly, and also sanctions to address a wide-ranging objective in the direction of improving features of plant and their yields. The last few years have introduced a number of unique genome editing systems, including transcription activator-like effector nucleases, zinc finger nucleases, and miRNA-regulated clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). Genome editing systems have helped in the transformation of metabolic engineering, allowing researchers to modify biosynthetic pathways of different secondary metabolites. Given the growing relevance of editing genomes in plant research, the exciting novel methods are briefly reviewed in this chapter. Also, this chapter highlights recent discoveries on the CRISPR-based modification of natural products in different medicinal plants.