Transcription Activator-like Effector Research Articles

Applications of Genome Editing in Yeast with an Example of Tup1 Mutants Construction

Genome editing, which allows for the artificial modification of genes in organisms, has received significant attention in recent years. Currently, various genome editing tools have been developed based on different principles. ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases), and CRISPR-Cas9 are the three genome editing technologies that are predominantly utilized. This article reviews the principles and characteristics of these three genome editing tools and summarizes the advantages and disadvantages of each of them accordingly. Genome editing is also playing an increasingly important role in yeast, opening up unforeseen applications for human beings. Tup1, a crucial transcriptional corepressor factor in yeast, is associated with multiple metabolic pathways and plays a key role in gene expression regulation. Currently, our understanding of how Tup1 regulates gene transcription remains limited. To gain a better understanding of the detailed mechanisms of Tup1, genome editing can be utilized to deepen our knowledge of its function. Therefore, this article proposes a method that utilizes CRISPR-Cas9 to construct mutant variants of Tup1 in yeast. The main process of this method involves introducing plasmids containing artificially designed sgRNA and Cas9 protein sequences into yeast cells, allowing them to express and edit the yeast Tup1 sequence, ultimately generating yeast Tup1 mutants. This method allows for the efficient construction of engineered Tup1 mutants, facilitating further research on Tup1.

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.

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.

Improved Genome Editing in the Ascidian Ciona with CRISPR/Cas9 and TALEN.

The ascidian Ciona intestinalis type A (or Ciona robusta) is an important organism for elucidating the mechanisms that make the chordate body plan. CRISPR/Cas9 and TAL effector nuclease (TALEN) are widely used to quickly address genetic functions in Ciona. Our previously reported method of CRISPR/Cas9-mediated mutagenesis in this animal has inferior mutation rates compared to those of TALENs. We here describe an updated way to effectively mutate genes with CRISPR/Cas9 in Ciona. Although the construction of TALENs is much more laborious than that of CRISPR/Cas9, this technique is useful for tissue-specific knockouts that are not easy even by the optimized CRISPR/Cas9 method.

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.

The Complete Genome Resource of Xanthomonas oryzae pv. oryzae CIX2779 Includes the First Sequence of a Plasmid for an African Representative of This Rice Pathogen.

The bacterial plant pathogen Xanthomonas oryzae pv. oryzae is responsible for the foliar rice bacterial blight disease. Genetically contrasted, continent-specific, sublineages of this species can cause important damages to rice production both in Asia and Africa. We report on the genome of the CIX2779 strain of this pathogen, previously named NAI1 and originating from Niger. Oxford Nanopore long reads assembly and Illumina short reads polishing produced a genome sequence composed of a 4,725,792-bp circular chromosome and a 39,798-bp-long circular plasmid designated pCIX2779_1. The chromosome structure and base-level sequence are highly related to reference strains of African X. oryzae pv. oryzae and encode identical transcription activator-like effectors for virulence. Importantly, our in silico analysis strongly indicates that pCIX2779_1 is a genuine conjugative plasmid, the first indigenous one sequenced from an African strain of the X. oryzae species. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

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.

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.

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.

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.

Epigenetic Manipulation of Transposable and Repetitive Elements.

This protocol describes methods to design, assemble, and validate tools for targeted activation or repression of single-copy and multi-copy genes, including repetitive and transposable elements. It uses transcription activator-like effector (TALE) technology combined with VP64 activator or Kruppel-associated box (KRAB) repressor, both of which are potent transcriptional regulators that modify the epigenetic state of endogenous DNA loci. This protocol has been successfully used to simultaneously modify expression patterns of thousands of LINE-1 transposable elements and satellite repeats, both in cell culture model systems and in preimplantation mouse embryos.

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.

Editing of the rice importin gene IMPα1b results in sequestration of TAL effectors from plant cell nuclei

Transcription activator-like effectors (TALEs) produced by plant pathogenic bacteria mainly belonging to the genus Xanthomonas cause plant diseases through activation of host susceptibility genes in plant cell nuclei. How TALEs enter plant cell nuclei was not clear until recent studies about PthXo1 and TALI, two TALEs produced by Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc), the rice (Oryza sativa) pathogens that cause bacterial blight and bacterial leaf streak, respectively. Here, we report that rice importin IMPα1b serves as a nuclear transport receptor in rice plants to facilitate the nuclear import of PthXo1 and TALI from Xoo and Xoc, respectively. While wild-type (WT) rice plants support the nuclear import of PthXo1 and TALI, nuclear trafficking is defective in OsIMPα1b loss-of-function mutants generated by clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated 9 (Cas9)-based gene editing. In the edited plants infected by Xoo, OsIMPα1b sequesters PthXo1 from the nucleus, the PthXo1-targeted rice susceptibility gene is no longer activated, and bacterial virulence and blight disease are alleviated as a result. In the edited plants infected by Xoc, OsIMPα1b sequesters TALI from the nucleus, the role of TALI in suppressing rice defense responses is nullified, and rice defense responses are in turn activated to inhibit bacterial virulence and alleviate bacterial leaf streak severity.

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.

Efficient multitool/multiplex gene engineering with TALE-BE

TALE base editors are a recent addition to the genome editing toolbox. These molecular tools are fusions of a transcription activator-like effector domain (TALE), split-DddA deaminase halves, and an uracil glycosylase inhibitor (UGI) that have the distinct ability to directly edit double strand DNA, converting a cytosine (C) to a thymine (T). To dissect the editing rules of TALE-BE, we combined the screening of dozens of TALE-BE targeting nuclear genomic loci with a medium/high throughput strategy based on precise knock-in of TALE-BE target site collections into the cell genome. This latter approach allowed us to gain in depth insight of the editing rules in cellulo, while excluding confounding factors such as epigenetic and microenvironmental differences among different genomic loci. Using the knowledge gained, we designed TALE-BE targeting CD52 and achieved very high frequency of gene knock-out (up to 80% of phenotypic CD52 knock out). We further demonstrated that TALE-BE generate only insignificant levels of Indels and byproducts. Finally, we combined two molecular tools, a TALE-BE and a TALEN, for multiplex genome engineering, generating high levels of double gene knock-out (∼75%) without creation of translocations between the two targeted sites.

Xanthomonas Infection Transforms the Apoplast into an Accessible and Habitable Niche for Salmonella enterica.

The physiology of plant hosts can be dramatically altered by phytopathogens. Xanthomonas hortorum pv. gardneri is one such pathogen that creates an aqueous niche within the leaf apoplast by manipulating the plant via the transcription activator-like effector AvrHah1. Simultaneous immigration of X. hortorum pv. gardneri and Salmonella enterica to healthy tomato leaves results in increased survival of S. enterica as Xanthomonas infection progresses. However, the fate of S. enterica following arrival on actively infected leaves has not been examined. We hypothesized that the water soaking caused by X. hortorum pv. gardneri could facilitate the ingression of S. enterica into the apoplast and that this environment would be conducive for growth. We found that an altered apoplast, abiotically water congested or Xanthomonas infected and water-soaked, enabled surface S. enterica to passively localize to the protective apoplast and facilitated migration of S. enterica to distal sites within the aqueous apoplast. avrHah1 contributed to the protection and migration of S. enterica early in X. hortorum pv. gardneri infection. Xanthomonas-infected apoplasts facilitated prolonged survival and promoted S. enterica replication compared to the case with healthy apoplasts. Access to an aqueous apoplast in general protects S. enterica from immediate exposure to irradiation, whereas the altered environment created by Xanthomonas infection provides growth-conducive conditions for S. enterica. Overall, we have characterized an ecological relationship in which host infection converts an unreachable niche to a habitable environment. IMPORTANCE Bacterial spot disease caused by Xanthomonas species devastates tomato production worldwide. Salmonellosis outbreaks from consumption of raw produce have been linked to the arrival of Salmonella enterica on crop plants in the field via contaminated irrigation water. Considering that Xanthomonas is difficult to eradicate, it is highly likely that S. enterica arrives on leaves precolonized by Xanthomonas with infection under way. Our study demonstrated that infection and disease fundamentally alter the leaf, resulting in redistribution and change in abundance of a phyllosphere bacterial member. These findings contribute to our understanding of how S. enterica manages to persist on leaf tissue despite lacking the ability to liberate nutrients from plant cells. More broadly, this study reveals a mechanism by which physiochemical changes to a host environment imposed by a plant pathogen can convert an uninhabitable leaf environment into a hospitable niche for selected epiphytic microbes.

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.