Gene Editing Technology Research Articles

CRISPR-Cas9 potential for identifying novel therapeutic targets in muscle-invasive bladder cancer.

Gene editing technologies help identify the genetic perturbations driving tumour initiation, growth, metastasis and resistance to therapeutics. This wealth of information highlights tumour complexity and is driving cancer research towards precision medicine approaches based on an individual's tumour genetics. Bladder cancer is the 11th most common cancer in the UK, with high rates of relapse and low survival rates in patients with muscle-invasive bladder cancer (MIBC). MIBC is highly heterogeneous and encompasses multiple molecular subtypes, each with different responses to therapeutics. This evidence highlights the need to identify innovative therapeutic targets to address the challenges posed by this heterogeneity. CRISPR-Cas9 technologies have been used to advance our understanding of MIBC and determine novel drug targets through the identification of drug resistance mechanisms, targetable cell-cycle regulators, and novel tumour suppressor and oncogenes. However, the use of these technologies in the clinic remains a substantial challenge and will require careful consideration of dosage, safety and ethics. CRISPR-Cas9 offers considerable potential for revolutionizing bladder cancer therapies, but substantial research is required for validation before these technologies can be used in the clinical setting.

Current Landscape of Various Techniques and Methods of Gene Therapy through CRISPR Cas9 along with its Pharmacological and Interventional Therapies in the Treatment of Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is frequently referred to as a "lifestyle illness". In 2000, India (31.7 million) had the greatest global prevalence of diabetes mellitus, followed by China (20.8 million), the United States (17.7 million), and other countries. In recent years, the treatment of gene therapy (T2DM) has attracted intensive interest. We aimed to critically review the literature on the various techniques and methods, which may be a possible novel approach through the gene therapy CRISPR Cas9 and some other gene editing techniques for T2DM. Interventional and pharmacological approaches for the treatment of T2DM were also included to identify novel therapies for its treatment. An extensive literature survey was done on databases like PubMed, Elsevier, Science Direct and Springer. It can be concluded from the study that recent advancements in gene-editing technologies, such as CRISPR Cas9, have opened new avenues for the development of novel therapeutic approaches for T2DM. CRISPR Cas9 is a powerful tool that enables precise and targeted modifications of the genome.

Rapid Answers: Gene-editing technology gives diagnostic tests a boost.

Rapid Answers: Gene-editing technology gives diagnostic tests a boost.

Insights into Prime Editing Technology: A Deep Dive into Fundamentals, Potentials, and Challenges.

As the most versatile and precise gene editing technology, prime editing (PE) can establish a durable cure for most human genetic disorders. Several generations of PE have been developed based on an editor machine or prime editing guide RNA (pegRNA) to achieve any kind of genetic correction. However, due to the early stage of development, PE complex elements need to be optimized for more efficient editing. Smart optimization of editor proteins as well as pegRNA has been contemplated by many researchers, but the universal PE machine's current shortcomings remain to be solved. The modification of PE elements, fine-tuning of the host genes, manipulation of epigenetics, and blockage of immune responses could be used to reach more efficient PE. Moreover, the host factors involved in the PE process, such as repair and innate immune system genes, have not been determined, and PE cell context dependency is still poorly understood. Regarding the large size of the PE elements, delivery is a significant challenge and the development of a universal viral or nonviral platform is still far from complete. PE versions with shortened variants of reverse transcriptase are still too large to fit in common viral vectors. Overall, PE faces challenges in optimization for efficiency, high context dependency during the cell cycling, and delivery due to the large size of elements. In addition, immune responses, unpredictability of outcomes, and off-target effects further limit its application, making it essential to address these issues for broader use in nonpersonalized gene editing. Besides, due to the limited number of suitable animal models and computational modeling, the prediction of the PE process remains challenging. In this review, the fundamentals of PE, including generations, potential, optimization, delivery, invivo barriers, and the future landscape of the technology are discussed.

Gene editing therapy for cardiovascular diseases.

The development of gene editing tools has been a significant area of research in the life sciences for nearly 30 years. These tools have been widely utilized in disease detection and mechanism research. In the new century, they have shown potential in addressing various scientific challenges and saving lives through gene editing therapies, particularly in combating cardiovascular disease (CVD). The rapid advancement of gene editing therapies has provided optimism for CVD patients. The progress of gene editing therapy for CVDs is a comprehensive reflection of the practical implementation of gene editing technology in both clinical and basic research settings, as well as the steady advancement of research and treatment of CVDs. This article provides an overview of the commonly utilized DNA-targeted gene editing tools developed thus far, with a specific focus on the application of these tools, particularly the clustered regularly interspaced short palindromic repeat/CRISPR-associated genes (Cas) (CRISPR/Cas) system, in CVD gene editing therapy. It also delves into the challenges and limitations of current gene editing therapies, while summarizing ongoing research and clinical trials related to CVD. The aim is to facilitate further exploration by relevant researchers by summarizing the successful applications of gene editing tools in the field of CVD.

Gene Editing Technologies: CRISPR/Cas9 and Beyond for Genetic Disease Therapy and Research

Gene editing technologies have revolutionized the field of genetic disease therapy and research. Among these, CRISPR/Cas9 stands out as a versatile tool for precisely targeting and modifying specific sequences within the genome. This paper provides an overview of CRISPR/Cas9 and other emerging gene editing technologies, discussing their potential applications in treating genetic diseases and advancing scientific research. Additionally, ethical considerations and challenges associated with gene editing are explored, along with future directions for this rapidly evolving field. Gene editing technologies have emerged as powerful tools in the field of genetic disease therapy and research, offering unprecedented precision and versatility in manipulating the genome. Among these technologies, CRISPR/Cas9 has garnered significant attention for its simplicity, efficiency, and accuracy in targeted gene modification.

Oxidative stress and metabolic process responses of Chlorella pyrenoidosa to nanoplastic exposure: Insights from integrated analysis of transcriptomics and metabolomics

Oxidative stress is a universal interpretation for the toxicity mechanism of nanoplastics to microalgae. However, there is a lack of deeper insight into the regulation mechanism in microalgae response to oxidative stress, thus affecting the prevention and control for nanoplastics hazard. The integrated analysis of transcriptomics and metabolomics was employed to investigate the mechanism for the oxidative stress response of Chlorella pyrenoidosa to nanoplastics and subsequently lock the according core pathways and driver genes induced. Results indicated that the linoleic acid metabolism, glycine (Gly)-serine (Ser)-threonine (Thr) metabolism, and arginine and proline metabolism pathways of C. pyrenoidosa were collectively involved in oxidative stress. The analysis of linoleic acid metabolism suggested that nanoplastics prompted algal cells to secrete more allelochemicals, thereby leading to destroy the immune system of cells. Gly-Ser-Thr metabolism and arginine and proline metabolism pathways were core pathways involved in algal regulation of cell membrane function and antioxidant system. Key genes, such as LOX2.3, SHM1, TRPA1, and proC1, are drivers of regulating the oxidative stress of algae cells. This investigation lays the foundation for future applications of gene editing technology to limit the hazards of nanoplastics on aquatic organism.

Recent advances in gene-editing approaches for tackling antibiotic resistance threats: a review.

Antibiotic resistance, a known global health challenge, involves the flow of bacteria and their genes among animals, humans, and their surrounding environment. It occurs when bacteria evolve and become less responsive to the drugs designated to kill them, making infections harder to treat. Despite several obstacles preventing the spread of genes and bacteria, pathogens regularly acquire novel resistance factors from other species, which reduces their ability to prevent and treat such bacterial infections. This issue requires coordinated efforts in healthcare, research, and public awareness to address its impact on human health worldwide. This review outlines how recent advances in gene editing technology, especially CRISPR/Cas9, unveil a breakthrough in combating antibiotic resistance. Our focus will remain on the relationship between CRISPR/cas9 and its impact on antibiotic resistance and its related infections. Moreover, the prospects of this new advanced research and the challenges of adopting these technologies against infections will be outlined by exploring its different derivatives and discussing their advantages and limitations over others, thereby providing a corresponding reference for the control and prevention of the spread of antibiotic resistance.

An aptamer-mediated base editing platform for simultaneous knockin and multiple gene knockout for allogeneic CAR-T cells generation

Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. Conventional gene editing platforms that rely on nuclease activity, such as Clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point™ base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared to the conventional CRISPR-Cas9 system. Site-specific knock-in of a chimeric antigen receptor (CAR) and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.

Murine models of erythroid 5ALA synthesis disorders and their conditional synthetic lethal dependency on pyridoxine

AbstractX-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP) are uncommon diseases caused by loss-of-function and gain-of-function mutations, respectively, in the erythroid form of 5-aminolevulinic acid synthetase (ALAS), ALAS2, which encodes the first enzyme in heme biosynthesis. A related congenital sideroblastic anemia (CSA) is due to mutations in SLC25A38 (solute carrier family 25 member A38), which supplies mitochondrial glycine for ALAS2 (SLC25A38–CSA). The lack of viable animal models has limited the studies on pathophysiology and development of therapies for these conditions. Here, using CRISPR-CAS9 gene editing technology, we have generated knockin mouse models that recapitulate the main features of XLSA and XLPP; and using conventional conditional gene targeting in embryonic stem cells, we also developed a faithful model of the SLC25A38-CSA. In addition to examining the phenotypes and natural history of each disease, we determine the effect of restriction or supplementation of dietary pyridoxine (vitamin B6), the essential cofactor of ALAS2, on the anemia and porphyria. In addition to the well-documented response of XLSA mutations to pyridoxine supplementation, we also demonstrate the relative insensitivity of the XLPP/EPP protoporphyrias, severe sensitivity of the XLSA models, and an extreme hypersensitivity of the SLC25A38-CSA model to pyridoxine deficiency, a phenotype that is not shared with another mouse hereditary anemia model, Hbbth3/+ β-thalassemia intermedia. Thus, in addition to generating animal models useful for examining the pathophysiology and treatment of these diseases, we have uncovered an unsuspected conditional synthetic lethality between the heme synthesis–related CSAs and pyridoxine deficiency. These findings have the potential to inform novel therapeutic paradigms for the treatment of these diseases.

Knock out aryl hydrocarbon receptor gene in adipose-derived mesenchymal stem cell line (PT-5006) by CRISPR/Cas9

Mesenchymal stem cells (MSCs) have garnered significant attention in biomedical studies due to their remarkable properties, such as self-renewal, differentiation into diverse cell types and immune responses. The proliferation and differentiation of MSCs are significantly influenced by the ligand-dependent transcription factor known as the aryl hydrocarbon receptor (AhR). In order to understand the roles of Ahr in adipose-derived MSCs (AD-MSCs), we disrupted the Ahr gene in this work using CRISPR/Cas9 gene editing technology. The gRNA/Cas9 dual vector and donor vector were introduced into the AD-MSC cell line (PT-5006). Green fluorescent protein (GFP) expression and puromycin resistance were used to identify the transfected cells. AhR-KO cells were cloned and confirmed by PCR and sequencing. By using RT-qPCR, the expression levels of AhR and the Ahr-related gene Cyp1B1 were investigated. The results showed that the knocked out of AhR using CRISPR/Cas9, resulting significantly decreased expression of 7.69-fold for AhR and 3.70-fold for Cyp1B1 in the cells. These cell clones and CRISPR/Cas9 vectors could be used as tools to investigate the functions of AhR in both AD-MSCs and other cell types.

Engineering strategies to safely drive CAR T-cells into the future.

Chimeric antigen receptor (CAR) T-cell therapy has proven a breakthrough in cancer treatment in the last decade, giving unprecedented results against hematological malignancies. All approved CAR T-cell products, as well as many being assessed in clinical trials, are generated using viral vectors to deploy the exogenous genetic material into T-cells. Viral vectors have a long-standing clinical history in gene delivery, and thus underwent iterations of optimization to improve their efficiency and safety. Nonetheless, their capacity to integrate semi-randomly into the host genome makes them potentially oncogenic via insertional mutagenesis and dysregulation of key cellular genes. Secondary cancers following CAR T-cell administration appear to be a rare adverse event. However several cases documented in the last few years put the spotlight on this issue, which might have been underestimated so far, given the relatively recent deployment of CAR T-cell therapies. Furthermore, the initial successes obtained in hematological malignancies have not yet been replicated in solid tumors. It is now clear that further enhancements are needed to allow CAR T-cells to increase long-term persistence, overcome exhaustion and cope with the immunosuppressive tumor microenvironment. To this aim, a variety of genomic engineering strategies are under evaluation, most relying on CRISPR/Cas9 or other gene editing technologies. These approaches are liable to introduce unintended, irreversible genomic alterations in the product cells. In the first part of this review, we will discuss the viral and non-viral approaches used for the generation of CAR T-cells, whereas in the second part we will focus on gene editing and non-gene editing T-cell engineering, with particular regard to advantages, limitations, and safety. Finally, we will critically analyze the different gene deployment and genomic engineering combinations, delineating strategies with a superior safety profile for the production of next-generation CAR T-cell.

Topoisomerase Inhibitors and PIM1 Kinase Inhibitors Improve Gene Editing Efficiency Mediated by CRISPR-Cas9 and Homology-Directed Repair.

The CRISPR-Cas9 system has emerged as the most prevalent gene editing technology due to its simplicity, high efficiency, and low cost. However, the homology-directed repair (HDR)-mediated gene knock-in in this system suffers from low efficiency, which limits its application in animal model preparation, gene therapy, and agricultural genetic improvement. Here, we report the design and optimization of a simple and efficient reporter-based assay to visualize and quantify HDR efficiency. Through random screening of a small molecule compound library, two groups of compounds, including the topoisomerase inhibitors and PIM1 kinase inhibitors, have been identified to promote HDR. Two representative compounds, etoposide and quercetagetin, also significantly enhance the efficiency of CRISPR-Cas9 and HDR-mediated gene knock-in in mouse embryos. Our study not only provides an assay to screen compounds that may facilitate HDR but also identifies useful tool compounds to facilitate the construction of genetically modified animal models with the CRISPR-Cas9 system.

Induced Pluripotent Stem Cells and CRISPR-Cas9 Innovations for Treating Alpha-1 Antitrypsin Deficiency and Glycogen Storage Diseases.

Human induced pluripotent stem cell (iPSC) and CRISPR-Cas9 gene-editing technologies have become powerful tools in disease modeling and treatment. By harnessing recent biotechnological advancements, this review aims to equip researchers and clinicians with a comprehensive and updated understanding of the evolving treatment landscape for metabolic and genetic disorders, highlighting how iPSCs provide a unique platform for detailed pathological modeling and pharmacological testing, driving forward precision medicine and drug discovery. Concurrently, CRISPR-Cas9 offers unprecedented precision in gene correction, presenting potential curative therapies that move beyond symptomatic treatment. Therefore, this review examines the transformative role of iPSC technology and CRISPR-Cas9 gene editing in addressing metabolic and genetic disorders such as alpha-1 antitrypsin deficiency (A1AD) and glycogen storage disease (GSD), which significantly impact liver and pulmonary health and pose substantial challenges in clinical management. In addition, this review discusses significant achievements alongside persistent challenges such as technical limitations, ethical concerns, and regulatory hurdles. Future directions, including innovations in gene-editing accuracy and therapeutic delivery systems, are emphasized for next-generation therapies that leverage the full potential of iPSC and CRISPR-Cas9 technologies.

Latest advancements in the development of new therapies for type 1 diabetes.

This study aims to explore new treatments for type 1 diabetes that could serve as an alternative or adjunct to insulin therapy. This is aliterature review based on asearch of relevant scientific articles in PubMed, Scopus, Google Scholar, and Cochrane Library databases. The scrutiny of publications revealed that the introduction of glucagon-like peptide-1 agonists into insulin therapy can improve disease control and reduce the frequency of hypoglycaemic episodes. While immune therapy is pathogenetically justified, its utility is limited in patients with recent onset of type 1 diabetes. It may, however, find application in prophylaxis in individuals at increased risk of developing this type of diabetes. Concurrently, stem cell therapy is under active investigation in clinical trials and has shown promise in reducing insulin dependence, improving β-cell function and controlling glucose levels. In addition, stem cells have demonstrated efficacy in treating complications of diabetes such as diabetic nephropathy, peripheral neuropathy and diabetic angiopathy. There is compelling evidence supporting the significant potential of gene-editing technology. Intravenous administration of T-regulatory cells, as one method of cell therapy, shows potential in stabilising the course of diabetes and slowing its progression. However, further research is warranted to confirm efficacy. While gene therapy holds promise, much of its research is currently in the preclinical stage. Further development of innovative therapies for type 1 diabetes has the potential to enhance the quality of life of patients, improve disease control and prevent the development of complications (Fig. 1, Ref. 54). Keywords: diabetes type 1, treatment, cell therapy, insulin, pancreatic β-cells.

Advancements in Hematopoietic Stem Cell Gene Therapy: A Journey of Progress for Viral Transduction.

Hematopoietic stem cell (HSC) transduction has undergone remarkable advancements in recent years, revolutionizing the landscape of gene therapy specifically for inherited hematologic disorders. The evolution of viral vector-based transduction technologies, including retroviral and lentiviral vectors, has significantly enhanced the efficiency and specificity of gene delivery to HSCs. Additionally, the emergence of small molecules acting as transduction enhancers has addressed critical barriers in HSC transduction, unlocking new possibilities for therapeutic intervention. Furthermore, the advent of gene editing technologies, notably CRISPR-Cas9, has empowered precise genome modification in HSCs, paving the way for targeted gene correction. These striking progresses have led to the clinical approval of medicinal products based on engineered HSCs with impressive therapeutic benefits for patients. This review provides a comprehensive overview of the collective progress in HSC transduction via viral vectors for gene therapy with a specific focus on transduction enhancers, highlighting the latest key developments, challenges, and future directions towards personalized and curative treatments.

CRISPR/Cas9-based engineered Escherichia coli biosensor for sensitive and specific detection of Cd(II) in drinking water

Cadmium (Cd) is a ubiquitous pollutant that poses a potential threat to human health. Monitoring Cd(II) in drinking water has significant implications for preventing potential threats of Cd(II) to human. However, the weak signal output and response to nontarget interference limit the detection of Cd(II) using bacterial biosensors. In this study, to enable sensitive and specific detection of Cd(II) in water, a stable whole-cell biosensor, K12-PMP-luxCDABE-△cysI, was constructed in a dual-promoter mode by fusing the mercury promoter Pmer, regulatory gene merR(m), and luciferase gene luxCDABE into the E.coli chromosome based on CRISPR/Cas9 gene editing technology. By knocking out the cadmium-resistance-gene cysI, the sensitivity of the biosensor to Cd(II) was further enhanced. The constructed E. coli biosensor K12-PMP-luxCDABE-△cysI exhibited good nonlinear responses to 0.005–2 mg/L Cd(II). Notably, among the three constructed E. coli biosensor, it exhibited the strongest fluorescence intensity, with the limit of detection meeting the allowable limit for Cd(II) in drinking water. Simultaneously, it could specifically detect Cd(II). Nontarget metal ions, such as Zn(II), Hg(II), and Pb(II), did not affect its performance. Furthermore, it exhibited superior performance in detecting Cd(II) in real drinking water samples by avoiding background interference, and showed excellent stability with the relative standard deviation under 5%. Thus, K12-PMP-luxCDABE-△cysI holds promise as a potential tool for the detection of Cd(II) in drinking water.

CRISPR/Cas Genome Editing in Legume Crops: Challenges and Opportunities: A Review

Legumes are an important source of protein and provide a health-rich diet for human beings. It contains essential amino acids. It mainly plays a significant role in soil enrichment. Due to their role in agriculture and human nutrition, scientists have made efforts to develop new traits. The genetic enhancement of legumes was achieved using traditional breeding over the years however, the progress is very slow. Recent developments in genome editing technologies, specifically CRISPR-Cas technology, have improved key agricultural traits in legumes and offer a wealth of opportunities for studying traits like improved seed nutrient content, enhancing productivity and resilience to biotic and abiotic stresses recently introduced in legumes. So far, the genome editing technology has been effectively used in various legume crops, mainly soybean, peanut, cowpea and chickpea. Still, the transformation and regeneration of other legumes have remained a significant hurdle to the implementation of gene editing. This review mainly highlights the use of different gene editing technologies in legumes, progress and updates of CRISPR/Cas9 tools in legumes and challenges of legume crops face during production.

CRISPR-CAS System: Current Applications and Future Prospect

The CRISPR-Cas system is a revolutionary technology that has transformed gene editing and has the potential to revolutionize many fields. It is a bacterial immune system that can be programmed to recognize and cleave specific DNA sequences using Cas proteins and guide RNAs. This technology has numerous biotechnological and bioengineering applications and is simpler to design with high targeting efficiency and multiplex editing capability compared to older gene editing technologies. This review aims to explore the general application, impact on molecular biology and medicine, limitations, and future potential of the CRISPR-Cas system. With its potential to revolutionize medicine, agriculture, and environmental science, the CRISPR-Cas system is a powerful gene editing tool that offers hope for a wide range of applications.

Enhancing grain shape, thermotolerance, and alkaline tolerance via Gγ protein manipulation in rice.

Our findings highlight a valuable breeding resource, demonstrating the potential to concurrently enhance grain shape, thermotolerance, and alkaline tolerance by manipulating Gγ protein in rice. Temperate Geng/Japonica (GJ) rice yields have improved significantly, bolstering global food security. However, GJ rice breeding faces challenges, including enhancing grain quality, ensuring stable yields at warmer temperatures, and utilizing alkaline land. In this study, we employed CRISPR/Cas9 gene-editing technology to knock out the GS3 locus in seven elite GJ varieties with superior yield performance. Yield component measurements revealed that GS3 knockout mutants consistently enhanced grain length and reduced plant height in diverse genetic backgrounds. The impact of GS3 on the grain number per panicle and setting rate depended on the genetic background. GS3 knockout did not affect milling quality and minimally altered protein and amylose content but notably influenced chalkiness-related traits. GS3 knockout indiscriminately improved heat and alkali stress tolerance in the GJ varieties studied. Transcriptome analysis indicated differential gene expression between the GS3 mutants and their wild-type counterparts, enriched in biological processes related to photosynthesis, photosystem II stabilization, and pathways associated with photosynthesis and cutin, suberine, and wax biosynthesis. Our findings highlight GS3 as a breeding resource for concurrently improving grain shape, thermotolerance, and alkaline tolerance through Gγ protein manipulation in rice.