Frontiers in genome editing最新文献

Base editing has revolutionized genome engineering by enabling precise single-nucleotide modifications without inducing double-strand breaks. As a powerful and efficient gene-editing tool, base editors (BEs) have been widely applied in various model organisms, including zebrafish (Danio rerio), to facilitate functional genomic studies and disease modeling. Zebrafish, with its genetic similarity to humans and rapid development, provides an excellent platform for testing and optimizing emerging base editing technologies. This review comprehensively explores the advancements of cytosine and adenine base editors in zebrafish, highlighting recent developments that enhance efficiency, specificity, and editing scope. We discuss novel base editor variants tailored for zebrafish applications, improvements in delivery strategies, and methodologies to minimize off-target effects. Furthermore, we compare base editing with other precision genome-editing technologies, such as prime editing and homology-directed repair, to underscore its advantages in achieving targeted mutations with high fidelity. By evaluating the expanding role of base editing in zebrafish, this review provides valuable insights into its potential for translational research, genetic disease modeling, and future therapeutic applications.

Throughout history, leguminous crops have contributed significantly to the human diet. Grain legumes have long been identified as a valuable nutritional source for humans. However, their significance extends beyond nutrition to global food security, reducing reliance on chemical fertilizers, improving soil health and increasing resilience to climate change. Recognizing their vital importance in nutrition and agricultural production, scientists have worked persistently to uncover new genetic traits in legumes, resulting in enhanced yields, improved nutritional value and increased stress tolerance. Recently, the availability of genomic resources for new traits in grain legume plants has greatly increased, laying the groundwork for the adoption of advanced breeding technologies. Gene editing has shown significant potential to improve crop outcomes. This review critically examines the latest developments in gene-editing techniques specific to major grain legumes, focusing on their application in enhancing legume crops with significant agronomic characteristics. The article also shows the potential advantages associated with these advancements. Over the years, advancements in technologies such as Transcription Activator-Like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9), and the more recent Prime Editing technique have significantly contributed to genetic enhancements. These innovations have improved nutritional and market traits, boosted farming incomes, and increased the accessibility of affordable nutritious food, particularly in developing nations. Studies show that CRISPR/Cas9 is the most extensively applied gene editing technology in grain legumes. The advent of this technology has transformed genetic modification by offering exceptional precision and efficiency. This progress has enabled the creation of grain legumes that are more resistant to climate change and enhanced with improved nutritional content. Our research highlights that soybeans have been the primary focus of CRISPR/Cas9 gene editing efforts, surpassing any other grain legume, unlocking significant potential for innovation and improvement. This article presents a scientometric analysis of bibliographic data from the Web of Science using VOSviewer. It highlights global research trends, emphasizing China's leading role in international collaborations, the prominence of soybean (Glycine max) in CRISPR/Cas9 studies, and the key researchers driving advancements in gene editing for food security.

Introduction: CRISPR/Cas9-edited induced pluripotent stem cells (iPSCs) are valuable research models for mechanistic studies. However, gene conversion between a gene-pseudogene pair that share high sequence identity and form direct repeats in proximity on the same chromosome can interfere with the precision of gene editing. Mutations in the human beta-glucocerebrosidase gene (GBA1) are associated with Gaucher disease, Parkinson's disease, and Lewy body dementia. During the creation of a GBA1 KO iPSC line, we detected about 70% gene conversion from its pseudogene GBAP1. These events maintained the reading frame and resulted from GBA1-specific cleavage by CRISPR/Cas9, without disrupting the GBA1 gene.

Method: To increase the percentage of alleles with out-of-frame indels for triggering nonsense-mediated decay of the GBA1 mRNA, we supplied the cells with two single-stranded oligodeoxynucleotide (ssODN) donors as homology-directed repair (HDR) templates.

Results: We demonstrate that HDR using the ssODN templates effectively competes with gene conversion and enabled biallelic KO clone isolation, whereas the nonallelic homologous recombination (NAHR)-based deletion rate remained the same.

Discussion: Here, we report a generalizable method to direct cellular DNA repair of double strand breaks at a target gene towards the HDR pathway using exogenous ssODN templates, allowing specific editing of one gene in a gene-pseudogene pair without disturbing the other.

The CRISPR system has transformed many research areas, including cancer and immunology, by providing a simple yet effective genome editing system. Its simplicity has facilitated large-scale experiments to assess gene functionality across diverse biological contexts, generating extensive datasets that boosted the development of computational methods and machine learning/artificial intelligence applications. Integrating CRISPR with single-cell technologies has further advanced our understanding of genome function and its role in many biological processes, providing unprecedented insights into human biology and disease mechanisms. This powerful combination has accelerated AI-driven analyses, enhancing disease diagnostics, risk prediction, and therapeutic innovations. This review provides a comprehensive overview of CRISPR-based genome editing systems, highlighting their advancements, current progress, challenges, and future opportunities, especially in cancer and immunology.

CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) has emerged as a transformative genome-editing tool with significant therapeutic potential for age-related diseases, including Alzheimer's disease, Parkinson's disease, cardiovascular disorders, and osteoporosis. This study presents a bibliometric analysis of CRISPR-Cas9 research in age-related diseases, identifying key contributors, major research hotspots, and critical technological advancements. While promising applications have been demonstrated in gene repair, functional regulation, and molecular interventions, significant barriers persist, including off-target effects, low delivery efficiency, and limited editing in non-dividing cells. Ethical concerns over germline editing and gaps in long-term safety data further complicate clinical translation. Future directions emphasize the development of high-precision Cas9 variants, homology-directed repair-independent tools, and efficient delivery systems, alongside the establishment of international regulatory frameworks and multicenter clinical trials. These efforts are essential to fully realize the potential of CRISPR-Cas9 in addressing the global health challenges of aging.

Rare monogenic disorders are caused by mutations in single genes and have an incidence rate of less than 0.5%. Due to their low prevalence, these diseases often attract limited research and commercial interest, leading to significant unmet medical needs. In a therapeutic landscape where treatments are targeted to manage symptoms, gene editing therapy emerges as a promising approach to craft curative and lasting treatments for these patients, often referred to as "one-and-done" therapeutics. CRISPR-dependent base editing enables the precise correction of genetic mutations by direct modification of DNA bases without creating potentially deleterious DNA double-strand breaks. Base editors combine a nickase version of Cas9 with cytosine or adenine deaminases to convert C·G to T·A and A·T to G·C, respectively. Together, cytosine (CBE) and adenine (ABE) base editors can theoretically correct ∼95% of pathogenic transition mutations cataloged in ClinVar. This mini-review explores the application of base editing as a therapeutic approach for rare monogenic disorders. It provides an overview of the state of gene therapies and a comprehensive compilation of preclinical studies using base editing to treat rare monogenic disorders. Key considerations for designing base editing-driven therapeutics are summarized in a user-friendly guide for researchers interested in applying this technology to a specific rare monogenic disorder. Finally, we discuss the prospects and challenges for bench-to-bedside translation of base editing therapies for rare monogenic disorders.

Large genetic engineering constructs often face limitations in DNA element addition or replacement due to lack of unique endonuclease recognition sites. Traditional restriction resistance methods can identify CRISPR-induced mutants efficiently, but CRISPR target sites rarely contain suitable restriction motifs. Here, we demonstrate the use of SpCas9 combined with custom synthesised sgRNAs to linearize large plasmid constructs, enabling DNA element incorporation via seamless cloning methods. Additionally, SpCas9 and custom sgRNAs were used to digest target gene amplicons for effective genotyping of CRISPR-edited mutants, allowing us to distinguish between wild-type, heterozygous, and biallelic variants. This approach provides a straightforward, highly flexible method for modifying large plasmid constructs and screening CRISPR-induced edits.

Climate change is a global concern for agriculture, food security, and human health. It affects several crops and causes drastic losses in yield, leading to severe disturbances in the global economy, environment, and community. The consequences on important staple crops, such as rice, maize, and wheat, will worsen and create food insecurity across the globe. Although various methods of trait improvements in crops are available and are being used, clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) mediated genome manipulation have opened a new avenue for functional genomics and crop improvement. This review will discuss the progression in crop improvement from conventional breeding methods to advanced genome editing techniques and how the CRISPR/Cas9 technology can be applied to enhance the tolerance of the main cereal crops (wheat, rice, and maize) against any harsh climates. CRISPR/Cas endonucleases and their derived genetic engineering tools possess high accuracy, versatile, more specific, and easy to design, leading to climate-smart or resilient crops to combat food insecurity and survive harsh environments. The CRISPR/Cas9-mediated genome editing approach has been applied to various crops to make them climate resilient. This review, supported by a bibliometric analysis of recent literature, highlights the potential target genes/traits and addresses the significance of gene editing technologies in tackling the vulnerable effects of climate change on major staple crops staple such as wheat, rice, and maize.

Climate change threatens global crop yield and food security due to rising temperatures, erratic rainfall, and increased abiotic stresses like drought, heat, and salinity. Gene editing technologies, including CRISPR/Cas9, base editors, and prime editors, offer precise tools for enhancing crop resilience. This review explores the mechanisms of these technologies and their applications in developing climate-resilient crops to address future challenges. While CRISPR/enables targeted modifications of plant DNA, the base editors allow for direct base conversion without inducing double-stranded breaks, and the prime editors enable precise insertions, deletions, and substitutions. By understanding and manipulating key regulator genes involved in stress responses, such as DREB, HSP, SOS, ERECTA, HsfA1, and NHX; crop tolerance can be enhanced against drought, heat, and salt stress. Gene editing can improve traits related to root development, water use efficiency, stress response pathways, heat shock response, photosynthesis, membrane stability, ion homeostasis, osmotic adjustment, and oxidative stress response. Advancements in gene editing technologies, integration with genomics, phenomics, artificial intelligence (AI)/machine learning (ML) hold great promise. However, challenges such as off-target effects, delivery methods, and regulatory barriers must be addressed. This review highlights the potential of gene editing to develop climate-resilient crops, contributing to food security and sustainable agriculture.

Wheat is cultivated across diverse global environments, and its productivity is significantly impacted by various biotic stresses, most importantly but not limited to rust diseases, Fusarium head blight, wheat blast, and powdery mildew. The genetic diversity of modern cultivars has been eroded by domestication and selection, increasing their vulnerability to biotic stress due to uniformity. The rapid spread of new highly virulent and aggressive pathogen strains has exacerbated this situation. Three strategies can be used for enhancing disease resistance through genome editing: introducing resistance (R) gene-mediated resistance, engineering nucleotide-binding leucine-rich repeat receptors (NLRs), and manipulating susceptibility (S) genes to stop pathogens from exploiting these factors to support infection. Utilizing R gene-mediated resistance is the most common strategy for traditional breeding approaches, but the continuous evolution of pathogen effectors can eventually overcome this resistance. Moreover, modifying S genes can confer pleiotropic effects that hinder their use in agriculture. Enhancing disease resistance is paramount for sustainable wheat production and food security, and new tools and strategies are of great importance to the research community. The application of CRISPR-based genome editing provides promise to improve disease resistance, allowing access to a broader range of solutions beyond random mutagenesis or intraspecific variation, unlocking new ways to improve crops, and speeding up resistance breeding. Here, we first summarize the major disease resistance strategies in the context of important wheat diseases and their limitations. Next, we turn our attention to the powerful applications of genome editing technology in creating new wheat varieties against important wheat diseases.