Gene Editing Technology Research Articles

Recent Advances on Key Enzymes of Microbial Origin in the Lycopene Biosynthesis Pathway.

Lycopene is a common carotenoid found mainly in ripe red fruits and vegetables that is widely used in the food industry due to its characteristic color and health benefits. Microbial synthesis of lycopene is gradually replacing the traditional methods of plant extraction and chemical synthesis as a more economical and productive manufacturing strategy. The biosynthesis of lycopene is a typical multienzyme cascade reaction, and it is important to understand the characteristics of each key enzyme involved and how they are regulated. In this paper, the catalytic characteristics of the key enzymes involved in the lycopene biosynthesis pathway and related studies are first discussed in detail. Then, the strategies applied to the key enzymes of lycopene synthesis, including fusion proteins, enzyme screening, combinatorial engineering, CRISPR/Cas9-based gene editing, DNA assembly, and scaffolding technologies are purposefully illustrated and compared in terms of both traditional and emerging multienzyme regulatory strategies. Finally, future developments and regulatory options for multienzyme synthesis of lycopene and similar secondary metabolites are also discussed.

TRAF3 deficiency in MDCK cells improved sensitivity to the influenza A virus

Tumor necrosis factor receptor-associated factor 3 (TRAF3), an adaptor protein, has significant and varying effects on immunity depending on cell types. The role of TRAF3 in Madin-Darby Canine Kidney Epithelial (MDCK) cell resistance to influenza A virus (IVA) remains elusive. In the present study, CRISPR-Cas9 gene editing technology was used to construct the TRAF3 knockout MDCK cells (MDCK-TRAF3−/−). Hemagglutination assay, plaque assay, transcriptome, and quantitative real-time PCR were performed after IVA infection. The results showed that after IVA infection, HA titers and virus titers were promoted, interferon I-related pathways were significantly blocked, and transcription of several antiviral-related genes was significantly decreased in MDCK-TRAF3−/− cells. Thus, our study suggests that TRAF3 gene knockout reduced MDCK cell's resistance to IVA, thereby resulting in a promising way for IVA isolation and vaccine manufacturing.

Preclinical safety and biodistribution of CRISPR targeting SIV in non-human primates

In this study, we demonstrate the safety and utility of CRISPR-Cas9 gene editing technology for in vivo editing of proviral DNA in ART-treated, virally controlled simian immunodeficiency virus (SIV) infected rhesus macaques, an established model for HIV infection. EBT-001 is an AAV9-based vector delivering SaCas9 and dual guide RNAs designed to target multiple regions of the SIV genome: the viral LTRs, and the Gag gene. The results presented here demonstrate that a single IV inoculation of EBT-001 at each of 3 dose levels (1.4 × 1012, 1.4 × 1013 and 1.4 × 1014 genome copies/kg) resulted in broad and functional biodistribution of AAV9-EBT-001 to known tissue reservoirs of SIV. No off-target effects or abnormal pathology were observed, and animals returned to their normal body weight after receiving EBT-001. Importantly, the macaques that received the 2 highest doses of EBT-001 showed improved absolute lymphocyte counts as compared to antiretroviral-treated controls. Taken together, these results demonstrate safety, biodistribution, and in vivo proviral DNA editing following IV administration of EBT-001, supporting the further development of CRISPR-based gene editing as a potential therapeutic approach for HIV in humans.

Arabidopsis ecotype Ct-1, with its altered nitrate sensing ability, exhibits enhanced growth under low nitrate conditions in comparison to Col-0

To address the urgent need for sustainable solutions to the increased use of nitrogen fertilizers in agriculture, it is imperative to acquire an in-depth comprehension of the intricate interplay between plants and nitrogen. In this context, our research aimed to elucidate the molecular mechanism behind NO3- sensing/signaling in plants, which can enhance nitrogen utilization efficiency. Previous reports have revealed that the density and quantity of root hairs exhibit responsive behavior to varying levels of NO3-, while the precise molecular mechanisms governing these changes remain elusive. To further investigate this phenomenon, we specifically selected the Ct-1 ecotype, which manifested a greater abundance of root hairs compared to the Col-0 ecotype under conditions of low NO3-. Our investigations unveiled that the dissimilarities in the amino acid sequence of NRT1.1, a transceptor responsible for regulating nitrate signaling and transport, accounted for the observed variation in root hair numbers. These results suggest that NRT1.1 represents a promising target for gene editing technology, offering potential applications in enhancing the efficiency of nitrogen utilization in agricultural crops.

Modeling human brain rhabdoid tumor by inactivating tumor suppressor genes in induced pluripotent stem cells

Atypical teratoid/rhabdoid tumor (ATRT) is a rare childhood malignancy that originates in the central nervous system. Over ninety-five percent of ATRT patients have biallelic inactivation of the tumor suppressor gene SMARCB1. ATRT has no standard treatment, and a major limiting factor in therapeutic development is the lack of reliable ATRT models. We employed CRISPR/Cas9 gene-editing technology to knock out SMARCB1 and TP53 genes in human episomal induced pluripotent stem cells (Epi-iPSCs), followed by brief neural induction, to generate an ATRT-like model. The dual knockout Epi-iPSCs retained their stemness with the capacity to differentiate into three germ layers. High expression of OCT4 and NANOG in neurally induced knockout spheroids was comparable to that in two ATRT cell lines. Beta-catenin protein expression was higher in SMARCB1-deficient cells and spheroids than in normal Epi-iPSC-derived spheroids. Nucleophosmin, Osteopontin, and Ki-67 proteins were also expressed by the SMARCB1-deficient spheroids. In summary, the tumor model resembled embryonal features of ATRT and expressed ATRT biomarkers at mRNA and protein levels. Ribociclib, PTC-209, and the combination of clofilium tosylate and pazopanib decreased the viability of the ATRT-like cells. This disease modeling scheme may enable the establishment of individualized tumor models with patient-specific mutations and facilitate high-throughput drug testing.

Using Stems to Bear Fruit: Deciphering the Role of Alzheimer's Disease Risk Loci in Human-Induced Pluripotent Stem Cell-Derived Microglia.

Alzheimer's disease (AD) is the most common neurodegenerative disorder globally. In people aged 65 and older, it is estimated that 1 in 9 currently live with the disease. With aging being the greatest risk factor for disease onset, the physiological, social and economic burden continues to rise. Thus, AD remains a public health priority. Since 2007, genome-wide association studies (GWAS) have identified over 80 genomic loci with variants associated with increased AD risk. Although some variants are beginning to be characterized, the effects of many risk loci remain to be elucidated. One advancement which may help provide a patient-focused approach to tackle this issue is the application of gene editing technology and human-induced pluripotent stem cells (hiPSCs). The relatively non-invasive acquisition of cells from patients with known AD risk loci may provide important insights into the pathological role of these risk variants. Of the risk genes identified, many have been associated with the immune system, including ABCA7, CLU, MEF2C, PICALM and TREM2-genes known to be highly expressed in microglia. This review will detail the potential of using hiPSC-derived microglia to help clarify the role of immune-associated genetic risk variants in AD.

Molecular breeding of livestock for disease resistance

Animal infectious diseases pose a significant threat to the global agriculture and biomedicine industries, leading to significant economic losses and public health risks. The emergence and spread of viral infections such as African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), and avian influenza virus (AIV) have highlighted the need for innovative approaches to develop resilient and disease-resistant animal populations. Gene editing technologies, such as CRISPR/Cas9, offer a promising avenue for generating animals with enhanced disease resistance. This review summarizes recent advances in molecular breeding strategies for generating disease-resistant animals, focusing on the development of disease-resistant livestock. We also highlight the potential applications of genome-wide CRISPR/Cas9 library screening and base editors in producing precise gene modified livestock for disease resistance in the future. Overall, gene editing technologies have the potential to revolutionize animal breeding and improve animal health and welfare.

Human Epidermal Growth Factor Receptor 2 Targeting Specific T Cells Immunotherapy for Gastric Cancer

The current traditional treatment methods have no apparent advantages in further improving the survival rate of gastric cancer patients. With the rapid development of molecular biology and gene editing technology, a new type of antitumor treatment model - human epidermal growth factor receptor 2 (HER-2), targets chimeric antigen receptor T (CAR-T) cells. Immunotherapy has shown great potential and therapeutic prospects. CAR-T cell immunotherapy has made significant progress in the treatment of solid tumors. However, due to the high heterogeneity of gastric cancer and the complex tumor microenvironment, the immune escape of tumors is caused, and the treatment of gastric cancer still faces many challenges. This article reviews the research progress of HER-2 targeting CAR-T cell immunotherapy for gastric cancer and hopes that it will become a safe and effective treatment for gastric cancer.

Abstract P1064: Gene Editing Technologies To Understand Knockout And Over Expression Of Circadian Rhythm Regulatory Genes In Human Derived Spheroid Model.

Introduction: Human induced pluripotent stem cells (hiPSCs) have the ability to produce a huge variety of terminally differentiated cell types that can be used for tissue engineering, drug research, and cell therapy. Using CRISPR/Cas9 gene-editing technologies, hiPSCs with knockout mutations for Per1, Per2, CIITA, B2M, N-Cadherin, Connexin43 and over expression of Connexin43 were created. Cardiomyocytes (CM) were created from hiPSCs, and spheroid culture was carried out as previously described. The molecular basis of circadian regulation of human induced pluripotent stem cells (hiPSCs) is still under investigation. Methods: From hiPSC CMs, we created and described a 3D spheroid culture. We assessed the effectiveness of spheroid transplantation for cardiac healing in immunecompromised mice and pigs. We investigated border zone macrophage markers, border zone vascular density, and cell proliferation in myocardial infarction after spheroid transplantation. We used pedigree analysis and 20 years of family health diagnostics data to analyze the clinical consequences of type 2 diabetes medications among metformin combo patients in big extended family (N=30).. Results: TNF-Alpha, IL1-beta, NOS2, and IL-6 relative expression were shown to be lower in the KO group than in the WT group. Significant engraftment of hiPSC-derived cardiac spheroids was seen up to 4 weeks after transplantation, according to histological examination. Arrhythmogenicity is significantly altered by the spheroid therapy. Ventricular tachycardia may be related to macrophage immunological regulation. To further understand, we looked at the effects of a medication combination of dapagliflozin and metformin in a large extended family, where 5 out of 30 members had type 2 diabetes. One patient experienced mildly tachycardic heart issues over the course of a 20-year medication treatment. Conclusion: Our study established the promising therapeutic potential of spheroids model from hiPSC differentiated CMs to understand Circadian rhythm gene regulation.

Advances in Synthetic Biology Techniques and Industrial Applications of Corynebacterium glutamicum

Corynebacterium glutamicum is a Gram-positive bacterium (non-spore-forming) that has been wildly used for amino acid production. Due to its stable protein secretion, low extracellular hydrolase activity, and non-toxicity, the application field of C. glutamicum has been greatly expanded. Currently, gene editing technology based on synthetic biology has great potential for synthetic biology research and genetic modification in C. glutamicum because of its ability to efficiently regulate the physiological and metabolic networks of the strain. Therefore, we summarize the gene editing tools and strategies of C. glutamicum from the aspects of genetic modification and expression elements, and we also describe the effects of gene editing techniques on a variety of products such as amino acids and their derivatives, recombinant proteins, and functional sugars, which provide a certain theoretical basis for the research on the modification of C. glutamicum strains and industrial applications. Finally, we prospect the design and industrial application of C. glutamicum genetic modification from multiple perspectives based on gene editing techniques.

SYST-33 GENERATION OF ALLOGENEIC CAR-T CIRCUMVENTS FUNCTIONAL DEFICITS IN PATIENT-DERIVED AUTOLOGOUS PRODUCT FOR GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults, with a poor prognosis despite aggressive standard of care. Chimeric antigen receptor T-cell (CAR-T) therapy has shown promising results in liquid malignancies, but clinical trials in GBM targeting various tumor antigens have not shown durable clinical benefit. While this may be attributable to various tumor-intrinsic immune evasion strategies characteristic of GBM, little work has assessed whether the issue is due to the quality of the CAR-T treatment itself. Currently, CAR-Ts for clinical studies are manufactured in an autologous setting wherein T-cells are extracted from patients, engineered ex-vivo, and subsequently re-infused back. However, peripheral T-cells taken from untreated GBM patients have demonstrated qualitative and functional deficits, which may contribute to suboptimal treatment outcomes. Thus, we aimed to establish whether CAR-Ts generated from GBM patients would show reduced efficacy in comparison to healthy donors using our previously validated CD133 CAR-T. In this work, we show pre-treatment exhaustion and reduced survival advantage in autologous, patient-derived CD133-targeting CAR-T cell products using an orthotopic xenograft model of human GBM. To overcome the functional and logistical considerations of autologous therapy, we additionally aimed to generate an “off-the-shelf” allogeneic CD133 CAR-T. Using CRISPR gene editing technology, we generated TCR-knockout CAR-T cells with comparable pre-clinical efficacy to our autologous models. Ultimately, this work highlights the need to reassess autologous CAR-T therapy for GBM, and consider allogeneic approaches as biologically-informed therapeutic alternatives.

Abstract P1038: Role Of Hypomorphic Notch1 Mutations In Cardiomyocyte Signaling And Function In Hypoplastic Left Heart Syndrome

Background: Congenital heart disease (CHD) affects is the most common birth defect and affects 1% of births in the United States. Hypoplastic left heart syndrome (HLHS) a complex CHD where left sided structures are malformed during embryonic development. Compound heterozygous mutations in NOTCH1, a developmental transmembrane protein have been implicated HLHS patients. However, the effect of hypomorphic NOTCH1 expression on cardiomyocyte development has not been fully characterized. Methods: Using HLHS patient derived induced pluripotent stem cells (iPSCs) with a compound heterozygous NOTCH1 expression and CRISPR-Cas9 gene editing technology we developed multiple iPSCs clones with hypomorphic NOTCH1 expression. We characterized expression of NOTCH1 downstream signaling molecules using transcriptomics of iPSCs and CMs. We further characterized mitochondrial structure, ATP production and sarcomere architecture of the resulting CMs. Lastly, we characterized calcium transients and contractility of the NOTCH1 hypomorphic CMs. Results: iPSC-CMs with hypomorphic NOTCH1 expression showed reduced pluripotency and decreased differentiation into cardiomyocytes. Transcriptomics studies showed that hypomorphic clones increasingly differentiated into fibroblasts and smooth muscle cells as compared to wild-type (WT) cells. Multiple signaling pathways associated mitochondrial function, sarcomeric organization, and cardiomyocyte development were dysregulated. Live cell imaging showed abnormal sarcomeric architecture and mitochondrial hyperfission as compared to WT cells. ATP production was also decreased as compared to WT. Lastly, calcium transients in hypomorphic CMs showed a decrease in time to peak and upslope which consequently lead to decreased contractility and relaxation rates of hypomorphic NOTCH1 CMs. Conclusion: Hypomorphic expression of NOTCH1 seen in HLHS affects multiple aspects of CM cytoskeletal architecture during CM development. Consequently, NOTCH1 hypomorphic iPSC-CMs have abnormal calcium transients and decreased contractility, likely contributing to the higher incidence of arrhythmia and heart failure seen in HLHS patients.

Electrochemical Affinity Biosensors: Pervasive Devices with Exciting Alliances and Horizons Ahead.

Electrochemical affinity biosensors are evolving at breakneck speed, strengthening and colonizing more and more niches and drawing unimaginable roadmaps that increasingly make them protagonists of our daily lives. They achieve this by combining their intrinsic attributes with those acquired by leveraging the significant advances that occurred in (nano)materials technology, bio(nano)materials and nature-inspired receptors, gene editing and amplification technologies, and signal detection and processing techniques. The aim of this Perspective is to provide, with the support of recent representative and illustrative literature, an updated and critical view of the repertoire of opportunities, innovations, and applications offered by electrochemical affinity biosensors fueled by the key alliances indicated. In addition, the imminent challenges that these biodevices must face and the new directions in which they are envisioned as key players are discussed.

Sex-biased genome-editing effects of CRISPR-Cas9 across cancer cells dependent on p53 status

The CRISPR-Cas9 system has emerged as the dominant technology for gene editing and clinical applications. One major concern is its off-target effect after the introduction of exogenous CRISPR-Cas9 into cells. Several previous studies have investigated either Cas9 alone or CRISPR-Cas9 interactions with p53. Here, we reanalyzed previously reported data of p53-associated Cas9 activities and observed large significant sex differences between p53-wildtype and p53-mutant cells. To expand the impact of this finding, we further examined all protein-coding genes for sex-specific dependencies in a large-scale CRISPR-Cas9 screening dataset from the DepMap project. We highlighted the p53-dependent sex bias of gene knockouts (including MYC, PIK3CA, KAT2B, KDM4E, SUV39H1, FANCB, TLR7, and APC2) across cancer types and potential mechanisms (mediated by transcriptional factors, including SOX9, FOXO4, LEF1, and RYBP) underlying this phenomenon. Our results suggest that the p53-dependent sex bias may need to be considered in future clinical applications of CRISPR-Cas9, especially in cancer.

Novel molecular mechanism of high ribonucleic acid yield in Saccharomyces pastorianus revealed by transcriptomics

Ribonucleic acid (RNA) and its degradation products find widespread application across various industries, including the food condiments sector. This study employed comparative transcriptomics to investigate the genetic mechanisms underlying RNA synthesis in the mutant Saccharomyces pastorianus strain G03H8, which exhibited a high RNA yield. To identify genetically engineered targets, single gene over-expression and CRISPR/Cas9 gene editing technology were utilized. The differential enrichment results revealed a close association between RNA metabolism and cellular processes such as the cell cycle, purine metabolism, nucleotide metabolism, and other metabolic pathways. Subsequently, we selected the top nine most promising genes to evaluate their impact on RNA synthesis. In these nine targets, upon comparing the RNA yield of the genetically engineered strains, G03-TAL1, G03-PGM2, G03-ΔPRS5, and G03-△DBP8, with the parent strain G03, significant improvements of 31.7%, 41.8%, 72.1%, and 46.7% were observed, respectively. Furthermore, these four genes were found to enhance the strain's growth and augment the pentose phosphate pathway, thus demonstrating the considerable potential for enhancing RNA yield. Notably, this study represents the first instance of identifying the upregulation of TAL1, PGM2, PRS5, and DBP8 as a means to increase RNA content in S. pastorianus. Consequently, our findings present novel genetic targets for enhancing RNA production and contribute to a deeper understanding of the mechanisms involved in high RNA synthesis in S. pastorianus.

Maturation and refinement of the maculae and foveae in the Anolis sagrei lizard

The fovea is a pit in the center of the macula, which is a region of the retina with a high concentration of photoreceptor cells, which accounts for a large degree of visual acuity in primates. The maturation of this primate visual acuity area is characterized by the shallowing and widening of the foveal pit, a decrease in the diameter of the rod-free zone, and an increase in photoreceptor cells packing after birth. Maturation occurs concurrently with progressing age, increasing eye size, and retinal length/area. These observations have led to the hypothesis that the maturation of the fovea might be a function of mechanical variables that remodel the retina. However, this has never been explored outside of primates. Here, we take advantage of the Anolis sagrei lizard, which has a bifoveated retina, to study maturation of the fovea and macula. Eyes were collected from male and female lizards–hatchling, 2-month, 4-month, 6-month, and adult. We found that Anolis maculae undergo a maturation process somewhat different than what has been observed in primates. Anole macular diameters actually increase in size and undergo minimal photoreceptor cell packing, possessing a near complete complement of these cells at the time of hatching. As the anole eye expands, foveal centers experience little change in overall retina cell density with most cell redistribution occurring at macular borders and peripheral retina areas. Gene editing technology has recently been developed in lizards; this study provides a baseline of normal retina maturation for future genetic manipulation studies in anoles.

Split-Cas9-based targeted gene editing and nanobody-mediated proteolysis-targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells.

Precise regulation of partial critical proteins in cancer cells, such as anti-apoptotic proteins, is one of the crucial strategies for treating cancer and discovering related molecular mechanisms. Still, it is also challenging in actual research and practice. The widely used CRISPR/Cas9-based gene editing technology and proteolysis-targeting chimeras (PROTACs) have played an essential role in regulating gene expression and protein function in cells. However, the accuracy and controllability of their targeting remain necessary. Construction of UMUC-3-EGFP stable transgenic cell lines using the Sleeping Beauty system, Flow cytometry, quantitative real-time PCR, western blot, fluorescence microplate reader and fluorescence inverted microscope analysis of EGFP intensity. Characterization of Survivin inhibition was done by using Annexin V-FITC/PI apoptosis, calcein/PI/DAPI cell viability/cytotoxicity assay, cloning formation assay and scratch assay. The cell-derived xenograft (CDX) model was constructed to assess the in vivo effects of reducing Survivin expression. Herein, we established a synergistic control platform that coordinated photoactivatable split-Cas9 targeted gene editing and light-induced protein degradation, on which the Survivin gene in the nucleus was controllably edited by blue light irradiation (named paCas9-Survivin) and simultaneously the Survivin protein in the cytoplasm was degraded precisely by a nanobody-mediated target (named paProtacL-Survivin). Meanwhile, in vitro experiments demonstrated that reducing Survivin expression could effectively promote apoptosis and decrease the proliferation and migration of bladder cancerous cells. Furthermore, the CDX model was constructed using UMUC-3 cell lines, results from animal studies indicated that both the paCas9-Survivin system and paProtacL-Survivin significantly inhibited tumour growth, with higher inhibition rates when combined. In short, the coordinated regulatory strategies and combinable technology platforms offer clear advantages in controllability and targeting, as well as an excellent reference value and universal applicability in controlling the fate of cancer cells through multi-level regulation of key intracellular factors.

Costa Rican consumer perceptions of gene-editing

Costa Rica's rice production, a large determinant of the country's food security, is being negatively impacted by frequently increasing periods of intense drought. Costa Rican scientists have applied CRISPR/Cas9 to develop drought resistant rice varieties they believe the country's rice producers could benefit from. However, would Costa Ricans consume gene edited rice or products derived from this crop? A three-part, 26-question survey administered in-person to 1096 Costa Ricans uncovers their attitudes, knowledge, and perceptions of gene editing technology and crops. Multiple regressions were built where the independent variables were age, gender, education level, and subjective economic situation. No statistically significant relationships were found in the regression coefficients. Moreover, the k-means procedure (cluster analysis) was used to categorize respondents according to their attitudes on the consumption of gene-edited foods: negative, neutral, and positive. Results show that overall, Costa Rican consumers are open to the application of gene editing in agriculture and would consider consuming products derived from the application of the technology. They are also open to gene editing technology being used to address human and animal health issues. However, Costa Rican consumers are not open to gene editing being used to “design” human traits. This study adds evidence to the emerging literature on the acceptance of gene-edited food. It also highlights the importance of informing societies of just how vulnerable agriculture, and therefore food security, is to the increasingly adverse effects of climate change.

Research progress in mitochondrial gene editing technology.

Mitochondrial DNA (mtDNA) mutations result in a variety of genetic diseases. As an emerging therapeutic method, mtDNA editing technology recognizes targets more based on the protein and less on the nucleic acid. Although the protein recognition type mtDNA editing technology represented by zinc finger nuclease technology, transcription activator like effector nuclease technology and base editing technology has made some progress, the disadvantages of complex recognition sequence design hinder further popularization. Gene editing based on nucleic acid recognition by the CRISPR system shows superiority due to the simple structure, easy design and modification. However, the lack of effective means to deliver nucleic acids into mitochondria limits application in the field of mtDNA editing. With the advances in the study of endogenous and exogenous import pathways and the deepening understanding of DNA repair mechanisms, growing evidence shows the feasibility of nucleic acid delivery and the broad application prospects of nucleic acid recognition type mtDNA editing technology. Based on the classification of recognition elements, this article summarizes the current principles and development of mitochondrial gene editing technology, and discusses its application prospects.

A critical review on therapeutic approaches of CRISPR-Cas9 in diabetes mellitus.

Diabetes mellitus (D.M.) is a common metabolic disorder caused mainly by combining two primary factors, which are (1) defects in insulin production by the pancreatic β-cells and (2) responsiveness of insulin-sensitive tissues towards insulin. Despite the rapid advancement in medicine to suppress elevated blood glucose levels (hyperglycemia) and insulin resistance associated with this hazard, a demand has undoubtedly emerged to find more effective and curative dimensions in therapeutic approaches against D.M. The administration of diabetes treatment that emphasizes insulin production and sensitivity may result in unfavorable side effects, reduced adherence, and potential treatment ineffectiveness. Recent progressions in genome editing technologies, for instance, in zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR-Cas)-associated nucleases, have greatly influenced the gene editing technology from concepts to clinical practices. Improvements in genome editing technologies have also opened up the possibility to target and modify specific genome sequences in a cell directly. CRISPR/Cas9 has proven effective in utilizing ex vivo gene editing in embryonic stem cells and stem cells derived from patients. This application has facilitated the exploration of pancreatic beta-cell development and function. Furthermore, CRISPR/Cas9 enables the creation of innovative animal models for diabetes and assesses the effectiveness of different therapeutic strategies in treating the condition. We, therefore, present a critical review of the therapeutic approaches of the genome editing tool CRISPR-Cas9 in treating D.M., discussing the challenges and limitations of implementing this technology.