Editing Techniques Research Articles

Establishing a straightforward I-SceI-mediated recombination one-plasmid system for efficient genome editing in P. putida KT2440.

Pseudomonas putida has become an increasingly important chassis for producing valuable bioproducts. This development is not least due to the ever-improving genetic toolbox, including gene and genome editing techniques. Here, we present a novel, one-plasmid design of a critical genetic tool, the pEMG/pSW system, guaranteeing one engineering cycle to be finalized in 3 days. The pEMG/pSW system proved in the last decade to be valuable for targeted genome engineering in Pseudomonas, as it enables the deletion of large regions of the genome, the integration of heterologous gene clusters or the targeted generation of point mutations. Here, to expedite genetic engineering, two alternative plasmids were constructed: (1) The sacB gene from Bacillus subtilis was integrated into the I-SceI expressing plasmid pSW-2 as a counterselection marker to accelerated plasmid curing; (2) double-strand break introducing gene I-sceI and sacB counterselection marker were integrated into the backbone of the original pEMG vector, named pEMG-RIS. The single plasmid of pEMG-RIS allows rapid genome editing despite the low transcriptional activity of a single copy of the I-SceI encoding gene. Here, the usability of the pEMG-RIS is shown in P. putida KT2440 by integrating an expression cassette including an msfGFP gene in 3 days. In addition, a large fragment of 12.1 kb was also integrated. In summary, we present an updated pEMG/pSW genome editing system that allows efficient and rapid genome editing in P. putida. All plasmids designed in this study will be available via the Addgene platform.

Direct parental CRISPR gene editing in the predatory bug Orius strigicollis, a biocontrol agent against small arthropods.

The predatory flower bug Orius strigicollis serves as a valuable biocontrol agent against small arthropods; however, its effectiveness can vary, especially when population establishment fails due to low prey/pest densities. A promising approach to improve the efficacy of O. strigicollis as a biocontrol agent is through gene editing. However, as females lay their eggs in plant tissue, the conventional embryo injection approach is challenging in this species. In this study, we aimed to develop an efficient and practical gene editing technique for O. strigicollis using direct parental CRISPR (DIPA-CRISPR). Female bugs at various postemergence stages received Cas9 ribonucleoprotein injections, with subsequent genotyping of their offspring (G0) using PCR and a heteroduplex mobility assay. We targeted the kynurenine 3-monooxygenase gene (cinnabar), pivotal for insect ommochrome pigment biosynthesis. Through experimental optimization, we achieved a peak gene editing efficiency of 52%, i.e., 52% of G0 progeny carried gene-edited alleles when injecting 1 day postemergence. Notably, some gene-edited G0 adults exhibited a red-eye mosaic phenotype, in contrast to the black-eyed wild type. Crossing experiments confirmed the heritability of the introduced mutations in the subsequent generation (G1), enabling the establishment of a cinnabar-knockout line with bright red eyes. We demonstrate that our DIPA-CRISPR gene editing method tailored for O. strigicollis is efficient and practical. Our findings highlight the potency of DIPA-CRISPR as a tool for O. strigicollis genetic engineering and suggest broader applications for enhancing other biocontrol agents. © 2024 Society of Chemical Industry.

Advancing crop disease resistance through genome editing: a promising approach for enhancing agricultural production.

Modern agriculture has encountered several challenges in achieving constant yield stability especially due to disease outbreaks and lack of long-term disease-resistant crop cultivars. In the past, disease outbreaks in economically important crops had a major impact on food security and the economy. On the other hand climate-driven emergence of new pathovars or changes in their host specificity further poses a serious threat to sustainable agriculture. At present, chemical-based control strategies are frequently used to control microbial pathogens and pests, but they have detrimental impact on the environment and also resulted in the development of resistant phyto-pathogens. As a replacement, cultivating engineered disease-resistant crops can help to minimize the negative impact of regular pesticides on agriculture and the environment. Although traditional breeding and genetic engineering have been instrumental in crop disease improvement but they have certain limitations such as labour intensity, time consumption, and low efficiency. In this regard, genome editing has emerged as one of the potential tools for improving disease resistance in crops by targeting multiple traits with more accuracy and efficiency. For instance, genome editing techniques, such as CRISPR/Cas9, CRISPR/Cas13, base editing, TALENs, ZFNs, and meganucleases, have proved successful in improving disease resistance in crops through targeted mutagenesis, gene knockouts, knockdowns, modifications, and activation of target genes. CRISPR/Cas9 is unique among these techniques because of its remarkable efficacy, low risk of off-target repercussions, and ease of use. Some primary targets for developing CRISPR-mediated disease-resistant crops are host-susceptibility genes (the S gene method), resistance genes (R genes) and pathogen genetic material that prevents their development, broad-spectrum disease resistance. The use of genome editing methods has the potential to notably ameliorate crop disease resistance and transform agricultural practices in the future. This review highlights the impact of phyto-pathogens on agricultural productivity. Next, we discussed the tools for improving disease resistance while focusing on genome editing. We provided an update on the accomplishments of genome editing, and its potential to improve crop disease resistance against bacterial, fungal and viral pathogens in different crop systems. Finally, we highlighted the future challenges of genome editing in different crop systems for enhancing disease resistance.

NASP gene contributes to autism by epigenetic dysregulation of neural and immune pathways

BackgroundEpigenetics makes substantial contribution to the aetiology of autism spectrum disorder (ASD) and may harbour a unique opportunity to prevent the development of ASD. We aimed to identify novel epigenetic...

FLI1 is associated with regulation of DNA methylation and megakaryocytic differentiation in FPDMM caused by a RUNX1 transactivation domain mutation

Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation, the DNA methylation status in FPDMM remains unknown, largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here, using genome editing techniques, we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks), consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs, with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1, an ETS family member, was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation, and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation.

The Rhetorical Construction of Introductory Viewing in The Cinematic Film

Research studies address many aspects of the rhetorical construction of opening scenes in cinematic films, and among these studies it is possible to understand the rhetorical construction of opening scenes. The film is distinguished by its exciting and complex opening scenes that use many rhetorical methods to attract the attention of viewers and deliver the intended message. Here are some points that can be part of the rhetorical structure of introductory scenes. Use of image and sound: Image and sound are used innovatively in introductory scenes in the film to powerfully convey thoughts and feelings. Innovative filming techniques and appropriate sound effects are used to create an exciting and interesting cinematic experience. Tension and suspense: The opening scenes in the film are scenes full of tension and suspense. Many visual and audio elements are used to create an atmosphere of tension and suspense, such as exciting music and dynamic cinematography. Cinematic composition: Cinematic composition is used elaborately in the opening scenes of the film to convey the story and events smoothly and coherently. Editing and transition techniques are used expertly to create a cohesive narrative flow. Symbolism and Visual Symbols: Symbolism and visual symbols are distinctively used in the opening scenes of the film to convey deep messages and hidden meanings. Colors and visual symbols are used to give the viewer character and reinforce the message.

Y chromosome shredding in Anopheles gambiae: Insight into the cellular dynamics of a novel synthetic sex ratio distorter.

Despite efforts to explore the genome of the malaria vector Anopheles gambiae, the Y chromosome of this species remains enigmatic. The large number of repetitive and heterochromatic DNA sequences makes the Y chromosome exceptionally difficult to fully assemble, hampering the progress of gene editing techniques and functional studies for this chromosome. In this study, we made use of a bioinformatic platform to identify Y-specific repetitive DNA sequences that served as a target site for a CRISPR/Cas9 system. The activity of Cas9 in the reproductive organs of males caused damage to Y-bearing sperm without affecting their fertility, leading to a strong female bias in the progeny. Cytological investigation allowed us to identify meiotic defects and investigate sperm selection in this new synthetic sex ratio distorter system. In addition, alternative promoters enable us to target the Y chromosome in specific tissues and developmental stages of male mosquitoes, enabling studies that shed light on the role of this chromosome in male gametogenesis. This work paves the way for further insight into the poorly characterised Y chromosome of Anopheles gambiae. Moreover, the sex distorter strain we have generated promises to be a valuable tool for the advancement of studies in the field of developmental biology, with the potential to support the progress of genetic strategies aimed at controlling malaria mosquitoes and other pest species.

A gene edited pig model for studying LGR5+ stem cells: implications for future applications in tissue regeneration and biomedical research.

Recent advancements in genome editing techniques, notably CRISPR-Cas9 and TALENs, have marked a transformative era in biomedical research, significantly enhancing our understanding of disease mechanisms and helping develop novel therapies. These technologies have been instrumental in creating precise animal models for use in stem cell research and regenerative medicine. For instance, we have developed a transgenic pig model to enable the investigation of LGR5-expressing cells. The model was designed to induce the expression of H2B-GFP under the regulatory control of the LGR5 promoter via CRISPR/Cas9-mediated gene knock-in. Notably, advancements in stem cell research have identified distinct subpopulations of LGR5-expressing cells within adult human, mouse, and pig tissues. LGR5, a leucine-rich repeat-containing G protein-coupled receptor, enhances WNT signaling and these LGR5+ subpopulations demonstrate varied roles and anatomical distributions, underscoring the necessity for suitable translational models. This transgenic pig model facilitates the tracking of LGR5-expressing cells and has provided valuable insights into the roles of these cells across different tissues and species. For instance, in pulmonary tissue, Lgr5+ cells in mice are predominantly located in alveolar compartments, driving alveolar differentiation of epithelial progenitors via Wnt pathway activation. In contrast, in pigs and humans, these cells are situated in a unique sub-basal position adjacent to the airway epithelium. In fetal stages a pattern of LGR5 expression during lung bud tip formation is evident in humans and pigs but is lacking in mice. Species differences with respect to LGR5 expression have also been observed in the skin, intestines, and cochlea further reinforcing the need for careful selection of appropriate translational animal models. This paper discusses the potential utility of the LGR5+ pig model in exploring the role of LGR5+ cells in tissue development and regeneration with the goal of translating these findings into human and animal clinical applications.

Therapeutic Potential of CRISPR/Cas in Hashimoto's Thyroiditis: A Comprehensive Review.

Hashimoto's thyroiditis (HT) is a commonly occurring illness of autoimmune endocrine origin. It is usually present in the pediatric age group along with other well-known diseases, such as type 1 insulin-dependent diabetes. The defining feature of this disease is the immune-- mediated attack on the thyroid gland resulting in the destruction of thyroid tissues and cells. Given that HT frequently affects family members, it is well-recognized that individuals are genetically predisposed to this disease. Patients with HT also display a significantly increased risk for several different cancers, justifying the eminent need for the development of therapies for managing and treating HT. Gene editing has made several advancements in the field of molecular biology and has turned out to become a promising approach to correct several autoimmune diseases. Currently, CRISPR/Cas, a nuclease-based editing technique, is publicized as a promising tool for curing several genetic diseases and cancers. However, very limited research has been conducted as of now on autoimmune disease management and cure via CRISPR/Cas technique. This review provides an account of the potential candidate genes associated with Hashimoto's thyroiditis, and only a few animal and human models have been generated via the CRISPR/Cas gene editing technique. Mouse models of autoimmune thyroiditis generated through the CRISPR/Cas gene editing technique by targeting the candidate genes will provide us with a deeper insight into the pathophysiology of HT and further pave the way for the immunomodulation of HT via gene editing.

A CRISPR-Cas9 Technique for Gene Editing in T Cells

A CRISPR-Cas9 Technique for Gene Editing in T Cells

A CRISPR-Cas9 Technique for Gene Editing in T Cells

A CRISPR-Cas9 Technique for Gene Editing in T Cells

Gene-Based Therapy for Brain Cancer: Promises and Challenges

Brain cancer, including gliomas and other malignant tumors, represents a devastating and challenging disease with limited treatment options and poor prognosis. Conventional therapies such as surgery, radiation, and chemotherapy have shown limited success, necessitating the exploration of alternative approaches. Gene-based therapy has emerged as a promising frontier in precision medicine, leveraging our growing understanding of the genetic and molecular drivers underlying brain tumor development and progression. This paper provides an overview of recent advancements in gene-based therapies for brain cancer, including gene editing techniques, gene silencing strategies, Immunotherapy, and more. Notably, these approaches aim to target specific oncogenes, tumor suppressor genes, and immune-related genes to inhibit tumor growth, induce apoptosis, and enhance the patient's immune response against cancer cells. Moreover, gene-based therapies offer the potential for personalized treatments tailored to individual patients based on their unique genetic profile, increasing treatment efficacy and minimizing adverse effects. While gene-based therapies have shown promising preclinical and early clinical results, challenges related to delivery, safety, and off-target effects remain to be addressed. This abstract emphasizes the urgent need for further research and large-scale clinical trials to establish the safety and long-term efficacy of gene-based therapies for brain cancer treatment. As the field continues to evolve, gene-based therapy holds great promise in reshaping the landscape of brain cancer management and providing hope for improved patient outcomes in the future.

CRISPR knockout of HRE sequences in the C9orf72 gene to treat ALS, FTD, and HDL2

Amyotrophic lateral sclerosis (ALS), familial frontotemporal dementia (FTD), and phenotypic Huntington syndrome-like disease (HDL2) have in common multiple hexanucleotide repeat expansions (HREs), in the C9orf72 gene. The C9orf72 protein aids in sending and receiving signals in multiple neurons. The C9orf72 protein is found primarily expressed in the brain and specific neurons, especially in the cerebral cortex. In ALS, FTD, and HDL2, the gene has a repeat sequence GGGGCC that is found in many patients with familial neurodegenerative conditions, resulting in the loss of function of many patients' motor neurons. The repeat sequence causes reduced production of the C9orf72 protein, called haploinsufficiency, and simultaneously a gain-of-function mutation by producing toxic dipeptide proteins, and excess RNA foci (RNA toxicity) in the brain. Different gene editing techniques in vitro and in vivo have shown the successful knockout of genes in the brain as well as on the C9orf72 gene and the connection to ALS, FTD, and Huntington-like syndrome in mice. CRISPR is a gene-editing tool used to target HREs. This review will focus on knocking down/out the HREs of the C9orf72 gene using various delivery strategies. Targeting the C9orf72 gene HREs, it would be expected that a decrease in protein aggregations that affect motor neurons, be able to prevent protein haploinsufficiency and prevent RNA toxicity, which are the main factors of these three neurodegenerative diseases. Using gene editing, treatment may be possible for patients with familial ALS, FTD, and HDL2 with the HRE sequences in the C9orf72 gene.

Establishment of a Secretory Protein-Inducible CRISPR/Cas9 System for Nosema bombycis in Insect Cells.

Gene editing techniques are widely and effectively used for the control of pathogens, but it is difficult to directly edit the genes of Microsporidia due to its unique spore wall structure. Innovative technologies and methods are urgently needed to break through this limitation of microsporidia therapies. Here, we establish a microsporidia-inducible gene editing system through core components of microsporidia secreted proteins, which could edit target genes after infection with microsporidia. We identified that Nosema bombycis NB29 is a secretory protein and found to interact with itself. The NB29-N3, which lacked the nuclear localization signal, was localized in the cytoplasm, and could be tracked into the nucleus after interacting with NB29-B. Furthermore, the gene editing system was constructed with the Cas9 protein expressed in fusion with the NB29-N3. The system could edit the exogenous gene EGFP and the endogenous gene BmRpn3 after overexpression of NB29 or infection with N. bombycis.

Advancing Nutritional Quality through Genomic Approaches for Biofortification in Cereal Crops: A Review

Hidden hunger, characterized by micronutrient deficiencies, remains a pervasive challenge in marginalized regions worldwide, where the main source of sustenance is cereal crops. This review study examines how improving the nutritional value of cereal crops through genetic "biofortification" could help fight hidden hunger. It focuses on the biochemical and genetic underpinnings of zinc, iron, and critical amino acid accumulation. While conventional breeding efforts have made significant contributions, the complex genetic nature of mineral content in grains presents challenges. In response, molecular techniques such as CRISPR/Cas genome editing offer promising solutions. The paper underscores the widespread prevalence of iron and zinc deficiencies, affecting over a large population. Children under five and pregnant and lactating women in developing countries are particularly vulnerable. Biofortification, defined as the breeding of staple food crops with elevated micronutrient levels, emerges as a cost-effective and sustainable strategy to improve health in resource-poor households. It discusses the transfer of genes and quantitative trait loci (QTLs) from wild and related species to cultivated wheat, emphasizing the need for marker-assisted selection and genomic selection to accelerate breeding progress. The emergence of CRISPR/Cas genome editing techniques in recent decades has revolutionized the field of plant breeding. The paper highlights the successful application of CRISPR/Cas9 in numerous cereal crops such as rice, wheat, maize and barley, to improve crop yields and nutritional content. It also explores the potential for precise base editing and gene expression modifications. However, challenges such as transformation efficiency, specific promoters, and ethical and regulatory concerns are also mentioned and discussed. In conclusion, genetic biofortification through CRISPR/Cas-mediated genome editing presents a promising avenue for alleviating hidden hunger in cereal-dependent regions.

CRISPR/Cas9 Technology: A Novel Approach to Obesity Research.

Gene editing technology, particularly Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has transformed medical research. As a newly developed genome editing technique, CRISPR technology has strongly assisted scientists in enriching their comprehension of the roles of individual genes and their influences on a vast spectrum of human malignancies. Despite considerable progress in elucidating obesity's molecular pathways, current anti-obesity medications fall short in effectiveness. A thorough understanding of the genetic foundations underlying various neurobiological pathways related to obesity, as well as the neuro-molecular mechanisms involved, is crucial for developing effective obesity treatments. Utilizing CRISPR-based technologies enables precise determination of the roles of genes that encode transcription factors or enzymes involved in processes, such as lipogenesis, lipolysis, glucose metabolism, and lipid storage within adipose tissue. This innovative approach allows for the targeted suppression or activation of genes regulating obesity, potentially leading to effective weight management strategies. In this review, we have provided a detailed overview of obesity's molecular genetics, the fundamentals of CRISPR/Cas9 technology, and how this technology contributes to the discovery and therapeutic targeting of new genes associated with obesity.

Antimicrobial resistance in the wild: Insights from epigenetics.

Spreading of bacterial and fungal strains that are resistant to antimicrobials poses a serious threat to the well-being of humans, animals, and plants. Antimicrobial resistance has been mainly investigated in clinical settings. However, throughout their evolutionary history microorganisms in the wild have encountered antimicrobial substances, forcing them to evolve strategies to combat antimicrobial action. It is well known that many of these strategies are based on genetic mechanisms, but these do not fully explain important aspects of the antimicrobial response such as the rapid development of resistance, reversible phenotypes, and hetero-resistance. Consequently, attention has turned toward epigenetic pathways that may offer additional insights into antimicrobial mechanisms. The aim of this review is to explore the epigenetic mechanisms that confer antimicrobial resistance, focusing on those that might be relevant for resistance in the wild. First, we examine the presence of antimicrobials in natural settings. Then we describe the documented epigenetic mechanisms in bacteria and fungi associated with antimicrobial resistance and discuss innovative epigenetic editing techniques to establish causality in this context. Finally, we discuss the relevance of these epigenetic mechanisms on the evolutionary dynamics of antimicrobial resistance in the wild, emphasizing the critical role of priming in the adaptation process. We underscore the necessity of incorporating non-genetic mechanisms into our understanding of antimicrobial resistance evolution. These mechanisms offer invaluable insights into the dynamics of antimicrobial adaptation within natural ecosystems.

Improving the Traits of Perilla frutescens (L.) Britt Using Gene Editing Technology.

Plant breeding has evolved significantly over time with the development of transformation and genome editing techniques. These new strategies help to improve desirable traits in plants. Perilla is a native oil crop grown in Korea. The leaves contain many secondary metabolites related to whitening, aging, antioxidants, and immunity, including rosmarinic acid, vitamin E, luteolin, anthocyanins, and beta-carotene. They are used as healthy and functional food ingredients. It is an industrially valuable cosmetics crop. In addition, perilla seeds are rich in polyunsaturated fatty acids, such as α-linolenic acid and linoleic acid. They are known to be effective in improving neutral lipids in the blood, improving blood circulation, and preventing dementia and cardiovascular diseases, making them excellent crops whose value can be increased through improved traits. This research will also benefit perilla seeds, which can increase their stock through various methods, such as the increased production of functional substances and improved productivity. Recently, significant attention has been paid to trait improvement research involving gene-editing technology. Among these strategies, CRISPR/Cas9 is highly adaptable, enabling accurate and efficient genome editing, targeted mutagenesis, gene knockouts, and the regulation of gene transcription. CRISPR/Cas9-based genome editing has enormous potential for improving perilla; however, the regulation of genome editing is still at an early stage. Therefore, this review summarizes the enhancement of perilla traits using genome editing technology and outlines future directions.

Advancements in Genetic Enhancement: CRISPR/Cas-Mediated Genome Editing in Leguminous Crops

Legumes play a crucial role in human nutrition and sustainable agriculture due to their high protein content and health-promoting phytochemicals. To accelerate genetic gain in yield, stress resilience, and nutritional quality, extensive efforts are underway. Recent advancements in genomic resources have paved the way for the application of cutting-edge breeding technologies like genomic selection and genome editing in legume crops. This review focuses on the latest advancements in CRISPR/Cas9-based gene editing technology, specifically tailored for improving traits in legume crops. While successful gene-editing methods have been established for crops like soybean, cowpea, and chickpea, challenges remain, particularly in overcoming the recalcitrance of certain legumes to gene transfer and regeneration in vitro. Strategies to enhance transformation and regeneration rates through modifications in culture methods and DNA delivery are discussed. Despite the potential benefits of gene editing in legume breeding, regulatory barriers pose significant challenges. A comparison of regulatory environments in different regions sheds light on the importance of favourable regulations and public acceptance for realizing CRISPR's potential in enhancing global food security. Additionally, leguminous crops are vital for their nutritional properties, nitrogen-fixing abilities, and contribution to sustainable agriculture. Traditional breeding methods focused on increasing yield, while recent advancements in genome editing techniques, notably CRISPR-Cas technology, have revolutionized the improvement of agronomic traits in legumes. This chapter provides insights into the application of genome editing tools for enhancing traits such as stress tolerance, yield, and seed content in grain legumes. Furthermore, it discusses the challenges and prospects associated with enhancing grain legumes using molecular breeding techniques. Genome editing has been successfully employed in diverse legumes, including model species like Medicago and widely cultivated crops such as soybean and chickpea, offering exciting opportunities for enhancing agronomic characteristics.

Advances in and Perspectives on Transgenic Technology and CRISPR-Cas9 Gene Editing in Broccoli.

Broccoli, a popular international Brassica oleracea crop, is an important export vegetable in China. Broccoli is not only rich in protein, vitamins, and minerals but also has anticancer and antiviral activities. Recently, an Agrobacterium-mediated transformation system has been established and optimized in broccoli, and transgenic transformation and CRISPR-Cas9 gene editing techniques have been applied to improve broccoli quality, postharvest shelf life, glucoraphanin accumulation, and disease and stress resistance, among other factors. The construction and application of genetic transformation technology systems have led to rapid development in broccoli worldwide, which is also good for functional gene identification of some potential traits in broccoli. This review comprehensively summarizes the progress in transgenic technology and CRISPR-Cas9 gene editing for broccoli over the past four decades. Moreover, it explores the potential for future integration of digital and smart technologies into genetic transformation processes, thus demonstrating the promise of even more sophisticated and targeted crop improvements. As the field continues to evolve, these innovations are expected to play a pivotal role in the sustainable production of broccoli and the enhancement of its nutritional and health benefits.