Development Of Gene Editing Technologies Research Articles

The Development of Gene Editing Technology and Controversial Issues: A Discussion

The fields of genetics and biomedicine stand to benefit greatly from the innovative technique known as gene editing technology, especially with the potential uses of CRISPR-Cas9.the creation of crops resistant to disease, the treatment of genetic illnesses, and the advancement of biomedical research in fields like sickle-cell anemia and rice gene editing are all made possible by CRISPR-Cas9's capacity to precisely pinpoint and edit specific genes. Despite being beneficial to humanity, the quick development of human gene editing technology has also brought up ethical, legal, societal, and technical issues. Its management, informed consent, privacy, and social equality are among the issues that require considerable attention.In an effort to give the scientific community something to think about, this article examines the state of gene editing today, its potential for the future, and the debates surrounding its application. It concentrates on the way the CRISPR/Cas system works, how CRISPER/Cas9 gene editing is used, and the range of problems it has brought up.

Hotspot Analysis and Frontier Exploration of Stem Cell Research in Intervertebral Disc Regeneration and Repair: A Bibliometric and Visualization Study

BackgroundStem cells have shown tremendous potential and vast prospects in the research of intervertebral disc (IVD) regeneration and repair, attracting considerable attention in recent years. In this study, a bibliometric analysis and visualization techniques were employed to probe and analyze the hotspots and frontiers of stem cell research in IVD regeneration and repair, aiming to provide valuable references and insights for further investigations. MethodsThis study utilized the Science Citation Index Expanded (SCI-Expanded) from the Web of Science Core Collection (WoSCC) database to retrieve and extract relevant literature records as research samples. Visual analysis tools such as VOSviewer 1.6.19, CiteSpace 6.2.R4, and bibliometric online analysis platforms were employed to construct scientific knowledge maps, providing a comprehensive and systematic exposition from various perspectives including collaboration networks, co-citation networks, and co-occurrence networks. ResultsA total of 1,075 relevant studies have been published in 303 journals by 4,181 authors from 1,198 institutions across 54 countries/regions. Over the past 20 years, the field of research has witnessed a significant growth in annual publications and citations. China and the United States have emerged as the primary participants and contributors, with the AO Research Institute Davos, Zhejiang University, and Tokai University being the top three leading research institutions. The most productive and highly cited author is Sakai D, who is regarded as a key leader in this research field. The journals with the highest number of publications and citations are Spine and Biomaterials, which are considered to be high-quality and authoritative core journals in this field. The current research focuses primarily on the sources and selection of stem cells, optimization of transplantation strategies, mechanisms of IVD regeneration, and the combined application of stem cells and biomaterials. However, there are still some challenges that need to be addressed, including post-transplantation stability, assessment of regenerative effects, and translation into clinical applications. Future research will concentrate on the diversity of stem cell sources, the application of novel biomaterials, personalized treatments, and the development of gene editing technologies, among other cutting-edge directions. ConclusionThis study utilized bibliometric analysis and visualization techniques to unveil the hotspots and frontiers in the research on stem cells for IVD regeneration and repair. These research findings provide essential guidance and references for further experimental design and clinical applications. However, additional experiments and clinical studies are still needed to address the challenges and difficulties faced in the field of IVD regeneration and repair, thus offering novel strategies and approaches for the treatment of IVD diseases.

BLOC1S1 promotes proliferation of goat spermatogonial stem cells

With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is believed to have anti-brucella potential. Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a BLOC1S1 overexpression vector was constructed by homologous recombination. The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes (PCNA, CDK2, CCND1), and EIF2S3Y, a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1. These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.

Genetic Databases and Gene Editing Tools for Enhancing Crop Resistance against Abiotic Stress.

Abiotic stresses extensively reduce agricultural crop production globally. Traditional breeding technology has been the fundamental approach used to cope with abiotic stresses. The development of gene editing technology for modifying genes responsible for the stresses and the related genetic networks has established the foundation for sustainable agriculture against environmental stress. Integrated approaches based on functional genomics and transcriptomics are now expanding the opportunities to elucidate the molecular mechanisms underlying abiotic stress responses. This review summarizes some of the features and weblinks of plant genome databases related to abiotic stress genes utilized for improving crops. The gene-editing tool based on clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has revolutionized stress tolerance research due to its simplicity, versatility, adaptability, flexibility, and broader applications. However, off-target and low cleavage efficiency hinder the successful application of CRISPR/Cas systems. Computational tools have been developed for designing highly competent gRNA with better cleavage efficiency. This powerful genome editing tool offers tremendous crop improvement opportunities, overcoming conventional breeding techniques' shortcomings. Furthermore, we also discuss the mechanistic insights of the CRISPR/Cas9-based genome editing technology. This review focused on the current advances in understanding plant species' abiotic stress response mechanism and applying the CRISPR/Cas system genome editing technology to develop crop resilience against drought, salinity, temperature, heavy metals, and herbicides.

Molecular breeding of farm animals through gene editing

The rapid development of biotechnology has promoted people's understanding of the biological functions of candidate genes for important economic traits in farm animals. Molecular breeding by gene editing has greatly revolutionized the breeding of farm animals. Through gene editing and embryo manipulation, breeds with designed economic or disease-resistant traits can be readily generated. Along with this fast progress, the safety assessment of gene-edited farm animals has attracted public and regulatory attention. This review summarizes the research progress of gene editing in farm animals, focusing on performance improvement, disease resistance, bioreactors, animal welfare, and environmental friendliness. The limitations and future development of gene editing technology in farm animal breeding are also discussed.

Research and prospect of CRISPR system in marine biological system

Gene editing is a widely used and promising technology in biology field. Its ability to precisely edit the endogenous genes of organisms is a major boost to the research process in the biological sciences. Among these gene editing technologies, CRISPR is a more effective system and it is in a wider range of applications. The CRISPR system has advantages over Zinc finger nuclease (ZFNs) technology and transcription activator-like effector nuclease (TALENs) technology, including higher efficiency, gentler construction and a decreased risk of off-target effects. Currently, the treatment of diseases, the creation of animal models, and the genetic breeding of plants all make extensive use of CRISPR technology. Despite its wide request in model construction, its application in marine organisms is just beginning. This review systematically introduces the classification, development of gene editing technology, and summarizes the current status and development prospects of the application of CRISPR system in marine organisms.

Non-small-molecule therapeutics for drug addiction: From pharmacokinetics modulating to synthetic biology

Drug addiction is a pervasive problem worldwide. It not only affects the abuser's life, but also poses a serious threat to public health. Accordingly, there is a strong demand for the development of novel and effective therapies for drug addiction. Considering that small-molecule drugs have only had limited success, there is great interest in developing alternative strategies that extend the reach of small-molecule-based therapies. The antibody-based trapping approach and the enzyme catalytic strategy have been considered as promising ways to reduce the euphoria of drug users by altering drug pharmacokinetics and decreasing drug concentrations in the central nervous system. However, these biological macromolecules are generally unstable and their in vivo half-lives are short. With the rapid development of gene editing technologies, it is possible to perform ex vivo gene therapy for the long-term and stable delivery of enzymes and other effector proteins, which could free abusers from frequent injections of therapeutics. By constructing programmed gene switches that regulate spatiotemporal gene expression in response to illicit drugs, this perspective proposed the concept of preventing drug addiction by the drug itself. This strategy enables the controlled release of therapeutic proteins in vivo and is expected to improve patient safety and compliance. This will open up new opportunities for next-generation medicine and hold great promise for expanding our ability to treat drug addiction.

Research on the development of genetically engineered xenogenic porcine skin and its application in the treatment of burn wounds

In the recent years, the shortage of allo-skin sources has resulted in great challenges for salvage of patients with large area severe burns. Although being similar to human skin in construction and function, the clinical application of xenogenic porcine skin in burn wound management is limited due to factors including immuno-rejection, porcine endogenous retroviruses infection, etc. With the development of gene editing technology, especially the emerge of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein-9 system, multiple target genes could be possibly edited at the same time, which will bring broad prospect for the application of xenogenic porcine skin in the treatment of burn wounds. The paper mainly discusses the development, the existed barrier, the strategies of gene modification/editing, and the applications and research of xenogenic porcine skin xenografts in the clinical treatment of burn wound.

Application of Gene Editing Technology in Resistance Breeding of Livestock.

As a new genetic engineering technology, gene editing can precisely modify the specific gene sequence of the organism’s genome. In the last 10 years, with the rapid development of gene editing technology, zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), and CRISPR/Cas9 systems have been applied to modify endogenous genes in organisms accurately. Now, gene editing technology has been used in mice, zebrafish, pigs, cattle, goats, sheep, rabbits, monkeys, and other species. Breeding for disease-resistance in agricultural animals tends to be a difficult task for traditional breeding, but gene editing technology has made this easier. In this work, we overview the development and application of gene editing technology in the resistance breeding of livestock. Also, we further discuss the prospects and outlooks of gene editing technology in disease-resistance breeding.

Current landscape of gene‐editing technology in biomedicine: Applications, advantages, challenges, and perspectives

The expanding genome editing toolbox has revolutionized life science research ranging from the bench to the bedside. These “molecular scissors” have offered us unprecedented abilities to manipulate nucleic acid sequences precisely in living cells from diverse species. Continued advances in genome editing exponentially broaden our knowledge of human genetics, epigenetics, molecular biology, and pathology. Currently, gene editing‐mediated therapies have led to impressive responses in patients with hematological diseases, including sickle cell disease and thalassemia. With the discovery of more efficient, precise and sophisticated gene‐editing tools, more therapeutic gene‐editing approaches will enter the clinic to treat various diseases, such as acquired immunodeficiency sydrome (AIDS), hematologic malignancies, and even severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection. These initial successes have spurred the further innovation and development of gene‐editing technology. In this review, we will introduce the architecture and mechanism of the current gene‐editing tools, including clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‐associated nuclease‐based tools and other protein‐based DNA targeting systems, and we summarize the meaningful applications of diverse technologies in preclinical studies, focusing on the establishment of disease models and diagnostic techniques. Finally, we provide a comprehensive overview of clinical information using gene‐editing therapeutics for treating various human diseases and emphasize the opportunities and challenges.

Application of CRISPR/Cas9 System in Establishing Large Animal Models.

The foundation for investigating the mechanisms of human diseases is the establishment of animal models, which are also widely used in agricultural industry, pharmaceutical applications, and clinical research. However, small animals such as rodents, which have been extensively used to create disease models, do not often fully mimic the key pathological changes and/or important symptoms of human disease. As a result, there is an emerging need to establish suitable large animal models that can recapitulate important phenotypes of human diseases for investigating pathogenesis and developing effective therapeutics. However, traditional genetic modification technologies used in establishing small animal models are difficultly applied for generating large animal models of human diseases. This difficulty has been overcome to a great extent by the recent development of gene editing technology, especially the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). In this review, we focus on the applications of CRISPR/Cas9 system to establishment of large animal models, including nonhuman primates, pigs, sheep, goats and dogs, for investigating disease pathogenesis and treatment. We also discuss the limitations of large animal models and possible solutions according to our current knowledge. Finally, we sum up the applications of the novel genome editing tool Base Editors (BEs) and its great potential for gene editing in large animals.

Gene Therapy for Rare Neurological Disorders.

There are over 7,000 diseases that are individually rare, but collectively affect millions of people worldwide. They are very commonly neurologic single-gene disorders. Recent advances in recombinant adeno-associated virus vectors have enabled breakthroughs, including US Food and Drug Administration (FDA)-approved gene therapies for inherited retinal dystrophy due to RPE65 mutation and spinal muscular atrophy. A range of other gene therapies for rare neurologic diseases are at various stages of development. Future development of gene editing technologies promises further to broaden the potential for more patients with these disorders to benefit from innovative therapies.

Primary Coenzyme Q10 Deficiency-7 and Pathogenic COQ4 Variants: Clinical Presentation, Biochemical Analyses, and Treatment.

Primary Coenzyme Q10 Deficiency-7 (COQ10D7) is a rare mitochondrial disorder caused by pathogenic COQ4 variants. In this review, we discuss the correlation of COQ4 genotypes, particularly the East Asian-specific c.370G > A variant, with the clinical presentations and therapeutic effectiveness of coenzyme Q10 supplementation from an exon-dependent perspective. Pathogenic COQ4 variants in exons 1–4 are associated with less life-threating presentations, late onset, responsiveness to CoQ10 therapy, and a relatively long lifespan. In contrast, pathogenic COQ4 variants in exons 5–7 are associated with early onset, unresponsiveness to CoQ10 therapy, and early death and are more fatal. Patients with the East Asian-specific c.370G > A variant displays intermediate disease severity with multi-systemic dysfunction, which is between that of the patients with variants in exons 1–4 and 5–7. The mechanism underlying this exon-dependent genotype-phenotype correlation may be associated with the structure and function of COQ4. Sex is shown unlikely to be associated with disease severity. While point-of-care high-throughput sequencing would be useful for the rapid diagnosis of pathogenic COQ4 variants, whereas biochemical analyses of the characteristic impairments in CoQ10 biosynthesis and mitochondrial respiratory chain activity, as well as the phenotypic rescue of the CoQ10 treatment, are necessary to confirm the pathogenicity of suspicious variants. In addition to CoQ10 derivatives, targeted drugs and gene therapy could be useful treatments for COQ10D7 depending on the in-depth functional investigations and the development of gene editing technologies. This review provides a fundamental reference for the sub-classification of COQ10D7 and aim to advance our knowledge of the pathogenesis, clinical diagnosis, and prognosis of this disease and possible interventions.

Repurposing the Homing Endonuclease I-SceI for Positive Selection and Development of Gene-Editing Technologies.

Prokaryote genomes encode diverse programmable DNA endonucleases with significant potential for biotechnology and gene editing. However, these endonucleases differ significantly in their properties, which must be screened and measured. While positive selection screens based on ccdB and barnase have been developed to evaluate such proteins, their high levels of toxicity make them challenging to use. Here, we develop and validate a more robust positive selection screen based on the homing endonuclease I-SceI. Candidate endonucleases target and cure the I-SceI expression plasmid preventing induction of I-SceI-mediated double strand DNA breaks that lead to cell death in E.coli. We validated this screen to measure the relative activity of SpCas9, xCas9, and eSpCas9 and demonstrated an ability to enrich for more active endonuclease variants from a mixed population. This system may be applied in high throughput to rapidly characterize novel programmable endonucleases and be adapted for directed evolution of endonuclease function.

Discovery in CRISPR-Cas9 system.

The 2020 Nobel Prize in Chemistry was awarded to the American scientist Jennifer A. Doudna and the French scientist Emmanuelle Charpentier, in recognition of their discovery in one of the greatest weapons in genetic technology: CRISPR-Cas9 gene scissors. The CRISPR-Cas system is a bacterial defense immune system against exogenous genetic material. Because the system can specifically recognize and cut DNA, this technology is widely used for precise editing of animal, plant, and microbial DNA. The discovery of CRISPR-Cas9 gene scissors enables the tedious and complicated cell gene editing work to be completed in a few weeks or even less, which has promoted the development of gene editing technology in various fields and brought revolutionary influence to the field of life sciences. At the same time, CRISPR gene editing technology has become one of the new therapies for tumors because of its large number of targets and relatively simple operation, and it also makes gene therapy possible. Although the technology still needs to solve technical problems such as off-target and promoter inefficiency, the CRISPR-Cas system will show its unique advantages in more fields with the continuous development of life science and basic medicine.

Optimized two-step electroporation process to achieve efficient nonviral-mediated gene insertion into primary T cells.

The development of gene editing technologies over the past years has allowed the precise and efficient insertion of transgenes into the genome of various cell types. Knock‐in approaches using homology‐directed repair and designer nucleases often rely on viral vectors, which can considerably impact the manufacturing cost and timeline of gene‐edited therapeutic products. An attractive alternative would be to use naked DNA as a repair template. However, such a strategy faces challenges such as cytotoxicity from double‐stranded DNA (dsDNA) to primary cells. Here, we sought to study the kinetics of transcription activator‐like effector nuclease (TALEN)‐mediated gene editing in primary T cells to improve nonviral gene knock‐in. Harnessing this knowledge, we developed a rapid and efficient gene insertion strategy based on either short single‐stranded oligonucleotides or large (2 Kb) linear naked dsDNA sequences. We demonstrated that a time‐controlled two‐step transfection protocol can substantially improve the efficiency of nonviral transgene integration in primary T cells. Using this approach, we achieved modification of up to ˜ 30% of T cells when inserting a chimeric antigen receptor (CAR) at the T‐cell receptor alpha constant region (TRAC) locus to generate ‘off‐the shelf’ CAR‐T cells.

The Role of Gut Microbiota in Tumor Immunotherapy.

Tumor immunotherapy is the fourth therapy after surgery, chemotherapy, and radiotherapy. It has made great breakthroughs in the treatment of some epithelial tumors and hematological tumors. However, its adverse reactions are common or even more serious, and the response rate in some solid tumors is not satisfactory. With the maturity of genomics and metabolomics technologies, the effect of intestinal microbiota in tumor development and treatment has gradually been recognized. The microbiota may affect tumor immunity by regulating the host immune system and tumor microenvironment. Some bacteria help fight tumors by activating immunity, while some bacteria mediate immunosuppression to help cancer cells escape from the immune system. More and more studies have revealed that the effects and complications of tumor immunotherapy are related to the composition of the gut microbiota. The composition of the intestinal microbiota that is sensitive to treatment or prone to adverse reactions has certain characteristics. These characteristics may be used as biomarkers to predict the prognosis of immunotherapy and may also be developed as “immune potentiators” to assist immunotherapy. Some clinical and preclinical studies have proved that microbial intervention, including microbial transplantation, can improve the sensitivity of immunotherapy or reduce adverse reactions to a certain extent. With the development of gene editing technology and nanotechnology, the design and development of engineered bacteria that contribute to immunotherapy has become a new research hotspot. Based on the relationship between the intestinal microbiota and immunotherapy, the correct mining of microbial information and the development of reasonable and feasible microbial intervention methods are expected to optimize tumor immunotherapy to a large extent and bring new breakthroughs in tumor treatment.

Potential of Mitochondrial Genome Editing for Human Fertility Health.

Mitochondrial DNA (mtDNA) encodes vital proteins and RNAs for the normal functioning of the mitochondria. Mutations in mtDNA leading to mitochondrial dysfunction are relevant to a large spectrum of diseases, including fertility disorders. Since mtDNA undergoes rather complex processes during gametogenesis and fertilization, clarification of the changes and functions of mtDNA and its essential impact on gamete quality and fertility during this process is of great significance. Thanks to the emergence and rapid development of gene editing technology, breakthroughs have been made in mitochondrial genome editing (MGE), offering great potential for the treatment of mtDNA-related diseases. In this review, we summarize the features of mitochondria and their unique genome, emphasizing their inheritance patterns; illustrate the role of mtDNA in gametogenesis and fertilization; and discuss potential therapies based on MGE as well as the outlook in this field.

Gene editing for the treatment of primary immunodeficiency disease

Gene editing is an advanced technique based on artificial nucleases and can precisely modify genome sequences. It has shown great application prospects in the field of medicine and has provided a new precision therapy for diseases. Primary immunodeficiency disease is a group of diseases caused by single gene mutation and characterized by recurrent and refractory infections, with an extremely high mortality rate. The application of gene editing has brought hope for curing these diseases. This article reviews the development of gene editing technology and briefly introduces the research and application of gene editing technology in primary immunodeficiency disease.

Technology Forecasting based on Topic Analysis and Social Network Analysis: A Case Study Focusing on Gene Editing Patents

Technology forecasting research is an indispensable means to master the development trend of technology and provide decision support for scientific research management. For patent documents, it does not provide keyword information, which makes the keyword based technology prediction method have some limitations in revealing research content and hidden topics in specific fields. In order to better reflect the technical information in the patent, this paper combines topic analysis and social network analysis to study the development trends of gene editing technology. First, the patent data of gene editing technology is collected from Derwent Innovations Index. Secondly, text mining software is adopted to draw a network graph of topic words, combined with Inverse Document Frequency (IDF) to construct a weighted adjacency matrix, and Social Network Analysis is used to obtain the degree of centrality of technical topic words. Finally, the technological trends of gene editing technology is explored by identifying the core themes of gene editing, highlighting themes and emerging themes, and some meaningful conclusions are also obtained. Based on the analysis results, this study finds that the development of gene editing technology is limited by factors such as ethics, law and cellular pollution. In addition, future research directions will be more inclined to optimize the safety and efficiency of gene editing technology.