Insights from Stem Cell and CRISPR-Based Therapies for Diabetes Mellitus: A Systematic Review

Ahmad M. Khalil ^* 

1. Department of Biological Sciences, Yarmouk University, Irbid, Jordan

* Correspondence: Ahmad M. Khalil 

Academic Editor: Masahiro Sato

Received: November 27, 2025 | Accepted: March 11, 2026 | Published: March 17, 2026

OBM Genetics 2026, Volume 10, Issue 1, doi:10.21926/obm.genet.2601329

Recommended citation: Khalil AM. Insights from Stem Cell and CRISPR-Based Therapies for Diabetes Mellitus: A Systematic Review. OBM Genetics 2026; 10(1): 329; doi:10.21926/obm.genet.2601329.

© 2026 by the authors. This is an open access article distributed under the conditions of the Creative Commons by Attribution License, which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is correctly cited.

1. Introduction

1.1 Molecular Aspects of Diabetes Mellitus

Diabetes mellitus (DM) is one of the oldest human diseases. It is a complicated, multi-factorial chronic metabolic disease characterized by hyperglycemia caused primarily by combining two main factors: (a) defects in insulin production due to a dysfunction or loss of pancreatic islet cells, especially insulin-secreting β-cells [1,2,3,4] leading to absolute insulin deficiency, and (b) responsiveness of insulin-sensitive tissues towards insulin [5].

There are two types of DM; Type 1 diabetes (T1D) and Type 2 diabetes (T2D). The T1D is distinguished by a failure in the pancreatic cells to construct insulin as a result of cytotoxic T-cell-based autoimmunity [4,5]. In T1D, the β-cells are destroyed by an impaired immune system [3,6]; thus, the insulin production stops. In contrast, in T2D, a defect in the insulin receptor causes decreased insulin production and insulin resistance [7]. The impairment of glucose-stimulated insulin secretion is not only evident in T2D but also during the early stages of T1D [8]. T2D is characterized by chronic hyperglycemia and lipid abnormalities (glucolipotoxicity) in peripheral tissues, insulin resistance, and poor glucose homeostasis. Other characteristics of T2D include induction of oxidative stress [9,10], endoplasmic reticulum (ER) stress, and mitochondrial dysfunction in βcells, eventually triggering apoptosis [11]. The ER stress response pathways can have harmful effects on pancreatic β-cells. Misfolding of proinsulin can result from excessive biosynthetic ER load, genetic predispositions, or proinsulin gene mutations, upsetting the ER folding environment [12]. Other reports [13] revealed immune-provoked processes in T2D, once thought to be primarily associated with T1D. A mechanistic comparison between the two types of DM is detailed in a recent review article [13].

It is well documented that genetics plays a key role, to varying degrees, in the development of all kinds of DM [14]. Identifying genetic markers is critical in diabetes research, as it links specific genes to the development of the disease. It is important to improve genetic screening to identify DM susceptibility genes, enabling early diagnosis, evaluation, and risk stratification. These markers can indicate abnormalities in blood sugar regulation, and finding them can offer a roadmap for tailored treatments. What makes this method particularly helpful is its potential to involve people early, which could alter the lives of those predisposed to diabetes.

In the past few decades, genome-wide association studies (GWAS) have provided revolutionary insights into the genetic basis of DM. Not surprisingly, most of the 403 genetic variations detected in GWAS to be associated with T2D [15,16] have been proven to influence β-cell function. A large number of genes associated with T2D have been identified, including single-nucleotide polymorphisms, deletions, insertions, and copy number variations in GWAS studies. This links the pathophysiology of the disease, and any possible use of the information in drug discovery is challenging.

Linkage analysis revealed that the major histocompatibility complex (human leukocyte antigen [HLA]) loci on chromosome 6 are a genetically vulnerable site for T1D [17]. The HLA region accounts for 40-50% of the genetic risk, while other genes like Insulin (INS) also contribute [17]. Although the genetic risk of T1D is most strongly linked to the HLA genes, more than 50 other genes or loci have been correlated with the disease, most of which are expressed in pancreatic β-cells [18]. Another investigation [19] identified genetic variations in the X-ray repair cross-complementing protein 4 （XRCC4) and the GLIS Family Zinc Finger 3 (GLIS3) genes that modulate β-cell reactions to unfolded protein stress, enhancing apoptosis and senescence. However, it is not easy to suppose causality from a mutual genetic variant associated with either T1D or T2D.

Variants in several genes, including those in Transcription Factor 7-Like 2 (TCF7L2), Solute Carrier Family 30 Member 8 (SLC30A8), and Melatonin Receptor 1B (MTNR1B), indicate a strong association with T2D risk [15]. Hyperglycemia is the property of T2D, associated with many candidate genes, e.g., Mitogen-Activated Protein Kinase 4 (MAPK4), Glucokinase Regulator (GCKR), Signal Transducer and Activator of Transcription 3 (STAT3), Suppressor of Cytokine Signaling 3 (SOCS3), Protein Tyrosine Phosphatase Non-Receptor Type 1 (PTPN1), and Phosphoenolpyruvate Carboxykinase (PEPCK) [15]. In addition, several recognized genes, such as ATP Binding Cassette Subfamily C Member 8 (ABCC8), TCF7L2, Solute Carrier Family 2 Member 2 (SLC2A2), and Calpain-10 (CAPN10), are known to affect blood insulin and glucose levels [20].

In the last few years, research has revealed the involvement of PANoptosis, a complex phenomenon produced by three cell death pathways: programmed apoptosis, necroptosis, and pyroptosis, in the development of DM and its complications [21]. Although the role of PANoptosis in DM and its complications is incompletely understood, non-coding RNAs (Long non-coding RNA [lncRNA], microRNA [miRNA], and Circular RNA [circRNA]), key regulators of gene expression, appear to have specific regulatory functions in the disease [21].

1.2 Global Impact of Diabetes

About 90% of diabetic patients worldwide are of T2D, making it the most frequent type [22]. Several reports [23,24] showed that as of 2030, more than 640 million individuals will be living with diabetes globally. The number is expected to exceed 783 million by 2045, with the fastest growth in low- and middle-income countries. The tendency toward sedentary living could be the major cause of the growing number of diabetic patients globally, which is anticipated to reach 366 million in the older population (>65 years) by 2030 [24]. Globally, T1D affects 8.75 million individuals, approximately 1.52 million patients under 20.

In the face of developments in healthcare and the move towards a healthy lifestyle, DM is not just an individual health problem; it carries considerable socio-economic implications. It presents as a worldwide health challenge [25]. The world will probably be struggling with one of the worst shots with this disease of “sweetness”.

1.3 Long-Term Consequences

Chronic hyperglycemia inevitably results in microvascular and macrovascular complications, resulting in organ dysfunction and/or failure, including neuropathy, nephropathy, retinopathy, peripheral vascular disease, morbidity, and mortality [7,9,24]. Research analyzing mortality data of patients with T1D in the general population showed that, on average, the life expectancy for people with T1D at a young age is 16 years less than that of those who do not have diabetes [24,26]. The lifespan of T1D patients was 10 years lesser than that of those who established the disease at an older age. Identifying these long-term health effects helps underline the urgency of effective interventions.

This review article aims to delve into current DM treatment with emphasis on the novel CRISPR-based genome editing (GE) technology for DM therapy. It concludes that this cutting-edge technology could lead to groundbreaking therapies for diabetes. It also discusses developments in the design and optimization of techniques to tackle the challenges of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated protein 9 (Cas9) system for translation of CRISPR/Cas9-based GE therapy into clinical practice.

The subsequent sections provide insight into ongoing research, the methodologies used in these studies, and future routes that promise to harness this potent GE tool.

2. Methods

A comprehensive survey of the literature focusing on the exploration of the recent advances in the CRISPR technology in the field of DM treatment was carried out using well-known databases such as the ‘Web of Science’, PubMed, and Google Scholar. The keywords used as search terms included ‘CRISPR’, ‘diabetes’, ‘gene editing’, ‘insulin therapy’, and islet β-cells. The snowball method was also used to extract other publications. Due to the exponential growth in the field and space limitations, only articles published in the last 20 years are included. Articles published in non-English languages, as well as those without full text availability, were excluded. The “Blind” collection/analysis of the data was the main criterion used to eliminate “bias” and ensure the quality of studies. After an in-depth reading of each study, we established the overall framework for this review, categorizing the literature into DM background, CRISPR overview, and nongenetic and genetic treatments. After vigorous screening and detailed evaluation, only 114 studies out of 351 were selected for data extraction and analysis (Figure 1). As for literature before 2014, only 4 publications providing a basic background were included. About 47% (54 out of 114) of the analyzed articles were published during the last 3 years (2023-2025). These papers focused on the genetic research and treatment of CRISPR-mediated GE technology. They were further subdivided into two main sections: one summarizes the latest advances in CRISPR techniques, and the other describes applications of the CRISPR system in editing DM-associated genes in pancreatic β-cells and in animal models.

Figure 1 PRISMA flow diagram: Presentation of the procedure of literature searching and selection with numbers of articles at each stage.

3. Results and Discussion

3.1 Treatment Strategies for DM

Ideally, the goal of effective DM management is to achieve optimal glycemic control, prevent or delay complications, reduce the potential consequences on other organs, and improve overall quality of life. All types of diabetes must be recognized and controlled at an early stage [27]. Unfortunately, this is not the case, as diabetes is usually diagnosed in its late stages. Therefore, DM patients must use multiple treatment modalities. Current therapeutic strategies often focus on symptom management rather than the disease’s root causes. These methods include diet, exercise, and medication, as well as insulin pumps and weight-loss surgery. However, the administration of diabetes treatment that stresses insulin production and sensitivity can result in undesirable side effects, decreased adherence, and potential therapeutic ineffectiveness. The potential GE strategies, particularly the CRISPR system, to reshape our approach to treating diabetes, are, therefore, an exciting development. The advent of GE technologies has altered the scene of biomedical research.

For the present review, we will divide the therapeutic strategies for DM into two main categories: I. The Nongenetic (Chemical) methods and II. The Genetic methods.

3.1.1 The Nongenetic (Chemical) Methods

In a recent comprehensive review article [10], the nongenetic DM therapeutic approaches have been subdivided into: (a) Pharmacological Drugs, (b) Antidiabetic Agents, and (c) Plant-derived Substances. In this regard, we add a fourth group (d) Monoclonal Antibodies. Each class acts on diverse physiological mechanisms to control blood glucose levels.

Pharmacological Drugs. This section includes traditional therapeutic drugs that control glucose levels but do not address the root causes. The key pharmacological subclasses for T2D treatment include: Biguanides (Metformin), Sulfonylureas (e.g., glibenclamide, glipizide), Thiazolidinediones (e.g., pioglitazone), and Alpha-glucosidase inhibitors (e.g., acarbose). Although these drugs are effective, many of them have limitations, such as hypoglycemia danger, weight gain, and declining efficiency with time [10].

Insulin remains vital for T1D patients and is often needed in advanced T2D cases. However, the current insulin injection treatments for diabetes, particularly T1D, are unable to control glucose fluctuations precisely, cannot offer a cure, and consequently, diabetic patients are required to take medications permanently. In addition, intensive insulin management comes with significant challenges and risks, such as gastrointestinal distress, tolerability issues, and weight gain, even when consumed in combination. Furthermore, pharmacological drugs frequently cause adverse complications of uncontrolled diabetes, including cardiovascular disease, TIA, nephropathy, and limb amputation [7]. Therefore, scientists are searching for novel diabetes treatments. Gene-editing technologies, such as CRISPR/Cas9, are promising therapeutic strategies for DM.

Antidiabetic Agents. Recent therapeutic advances have provided additional benefits beyond glucose reduction, such as cardioprotection, renal protection, and weight loss. These drugs include Dipeptidyl Peptidase 4 (DPP-4) inhibitors (e.g., sitagliptin, linagliptin) [10], Glucagon-Like Peptide-1 (GLP-1) receptor agonists (e.g., liraglutide and semaglutide) [28], and Sodium-Glucose Cotransporter-2 inhibitors (e.g., empagliflozin and dapagliflozin) [29].

DM patients have to be treated with insulin, insulin analogs, and non-insulin oral hypoglycemic medications such as finerenone, tirzepatide, and GLP-1 receptor inhibitors [7]. Tirzepatide is the most superior unimolecular dual Glucose-dependent Insulinotropic Polypeptide (GIP)/GLP-1 receptor co-agonist, to be used as a once-weekly subcutaneous injection in T2D. It has demonstrated remarkable efficacy in glycemic control, weight loss, and cardiometabolic improvement, making it a promising candidate for managing T2D comorbid with atherosclerosis. However, considerable interindividual variability in treatment response suggests a role for genetic determinants in influencing the effectiveness and toxicity of tirzepatide.

Raising GLP-1, a vital target hormone that accelerates insulin secretion, is one therapeutic strategy to reduce serum glucose levels and thus control metabolism in affected patients. GLP-1 receptor agonists are a group of medications that activate the GLP-1 receptor to manage T2D and obesity, rather than inhibit it. However, GLP-1 has a brief half-life owing to its extremely rapid breakdown by the enzyme DPP-4 [30]. To avoid GLP-1 degradation, various DPP-4 inhibitors, such as linagliptin, saxagliptin, sitagliptin, and vildagliptin, have been developed for insulin-mediated glucose control in T2D. In addition, traditional medications (administered orally or subcutaneously) are considered inefficient due to inherent pharmacological limitations and delivery method limitations [31]. This results in chemical instability, enzymatic hydrolysis, and subpar gastrointestinal absorption.

Regrettably, the permanent insulin treatment approach carries a substantial risk of severe hypoglycemia. Patients with T2D who primarily receive hypoglycemic drugs and insulin therapy for a long time should be particularly concerned about this risk, as they experience hypoglycemia circumstances just as frequently as individuals with T1D [32]. If severe hypoglycemia is not managed, it can cause morbidity and even mortality [24].

Plant-Derived Substances. Herbal medicine continues to be widely used in the management of diabetes, particularly in conventional systems. Plants like Garlic (Allium sativum), Green tea (Camellia sinensis), Cinnamon (Cinnamomum verum), and Ginger (Zingiber officinale), among others, have been traditionally employed as natural antidiabetic agents. In the future, the advancement of novel tactics like legume-derived peptides having anti-diabetic potential [33]. The bioactive compounds in these plants have shown substantial reductions in fasting glucose and glycated hemoglobin (HbA1c), as well as oxidative and inflammatory stress markers in both preclinical and clinical trials [10]. Although plant therapies may provide new hope for improved diabetes management, they often lack thorough standardization, regulatory approval, and long-term safety data. Interactions with traditional drugs also have to be carefully studied.

Having in mind that many of these medications have significant adverse side effects, such as hepatic impairment, and those dispensed parenterally daily may be burdensome, it is not uncommon that patients display low adherence [34]. Regardless of the reason, patient exhaustion and decreased adherence are connected with suboptimal inhibition of complications [35]. Since the anticipated decline in hypoglycemic episodes has not yet been realized, there is an urgent need for continued research to develop more innovative, more effective, and safer treatment solutions.

Monoclonal Antibodies. Monoclonal Antibodies (mAbs) have demonstrated substantial promise in the treatment of diabetes through immunobiological mechanisms [36]. Recent investigations have shown that mAbs targeting distinct pathways are critical for preventing and treating diabetes by modulating autoimmunity in T1D and controlling metabolic complications and insulin resistance in T2D [37]. In 2022, Teplizumab, a humanized CD3-directed mAb, was the first immunotherapy for T1D to be approved by the US Food and Drug Administration (FDA) for delaying the onset of Stage 3 T1D [38]. In the meantime, mAbs targeting hormone receptors or cytokines have revealed promising benefits in T2D treatment [39].

Despite the increasing body of evidence on mAbs in diabetes, there are still some limitations, such as lack of effective targets for β-cell regeneration [36]. In addition, the translatability of preclinical outcomes for T2D mAbs to human efficiency remains controversial. Moreover, challenges continue regarding the delivery efficacy and security profiles of mAbs therapeutics, resulting in insufficient local concentrations within the pancreas [36,37]. This necessitates higher doses that, as a result, increase the risks of infections and malignancy.

3.1.2 The Genetic Methods

By focusing on genetic issues, researchers are identifying pathways that may pave the way for innovative treatments. The ability to edit genes with accuracy enables scientists to explore the root causes of diabetes and ultimately find solutions that could alleviate or even cure the disease.

Gene Editing Technologies. Recent advances in GE technologies, for instance, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system have significantly influenced the GE proficiency from concepts to clinical practices [40]. The programmable nucleases (ZFNs, TALENs, CRISPR/Cas9) have enabled direct understanding of the growth, regeneration, insulin production, and secretion patterns of pancreatic β cells [4].

CRISPR/Cas9 is one of the most effective nuclease-mediated GE strategies, offering benefits over ZFNs and TALENs, including simplicity of target design, efficiency, and versatility. CRISPR is a unique GE tool that can be easily used in a broad range of applications. With the endorsement of the first CRISPR-mediated human therapy by the US FDA in late 2023 [41,42], CRISPR genome editing has entered a new era of human medicine. Based on the data in Figure 2, it’s clear that CRISPR-related publications have increased substantially compared to the other two strategies. The significant surge in publications underscores the considerable impact on driving research efforts toward leveraging CRISPR technology. Therefore, this review focuses on the current landscape of CRISPR systems, e.g., on Cas9 variants with advanced properties. This milestone marks the official entry of gene therapy into the CRISPR-Cas9 era.

Figure 2 Annual distribution and publication trends of the three major gene editing technologies. Data partially extracted from [43]. The number of articles in PubMed^® (https://pubmed.ncbi.nlm.nih.gov/, accessed on 5 September 2023) with search terms “CRISPR Cas9”, “TALEN”, and “zinc finger nuclease”.

CRISPR Basics: An Overview. CRISPR/Cas has evolved into a gene-editing tool that serves as a naturally occurring “acquired immune defense system” in most archaea and many bacteria against foreign DNA (plasmids or phages). The CRISPR/Cas systems have been categorized largely into two major classes: Class I and Class II [44]. The class I systems perform their function through multiprotein complexes, which makes the process complicated. In contrast, the Class II systems function via a single protein effector complex, which is comparatively easy to implement, fast, and has been widely investigated. Among all Type II systems, Cas9, Cas12a, and Cas13a have been the most extensively studied [45,46,47]. In addition to their programmable targeting ability, Cas12a and Cas13a have been researched for their nonspecific collateral activity, which enables them to cleave non-specific single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA) after the specific identification of the target (Table 1). These features have made the CRISPR/Cas system a highly promising technology for nucleic acid detection [48].

Table 1 Description and comparison of Cas variants: Cas9, Cas12, and Cas13.

Table 1 compares the three Cas variants: Cas9, Cas12a, and Cas13a. CRISPR/Cas9 consists of Cas9, which shows DNA cleavage activity, and a chimeric short guide RNA (sgRNA) that identifies the Cas9/sgRNA complex to a particular genomic site. The DNA cleavage capacity of Cas9 can be used for loss-of-function, and catalytically deactivated Cas9 can be combined with a variety of effector molecules, such as fluorescent proteins, gene regulatory factors, and deaminases, for cell imaging, gene expression regulation, and base editing [50].

As shown in Figure 3, CRISPR induces double-strand DNA breaks (DSBs) at specific genomic target sites. The induced DSBs are then repaired either by homologous recombination (HR) in the presence of donor DNA or by the error-prone non-homologous end-joining (NHEJ) mechanism. The choice of repair mechanism is heavily influenced by cell type and the availability of DNA templates. This process permits insertion/deletion (indels), or silencing of any desired gene [51]. The accuracy of this process depends on protospacer adjacent motif (PAM) identification and subsequent DNA interrogation, which must be entirely complementary to the sgRNA; if not, Cas9 will not dissociate the DNA strands and will continue to probe the DNA until it locates a perfect match [52].

Figure 3 Schematic representation of the basic structure and mechanism of action of the CRISPR main CRISPR/Cas systems that have been adopted for therapeutic applications. CRISPR Cas9 (A) versus CRISPR 12a (B) and CRISPR 13a (C). CRISPR/Cas9 (A) and CRISPR 12a (B), respectively, induce blunt and staggered double-stranded DNA breaks (DSBs) in gDNA. Error-prone nonhomologous end joining (NHEJ) repair causes an indel mutation (knockout), leading to a reading frame shift in the exon. The alternative DNA repair mechanism (Homology-directed repair; HDR) is more accurate but requires a homologous template to direct repair and the production of new DNA (Knock-in). (C) The CRISPR/Cas13a system consists of two parts: the Cas13a effector protein and a CRISPR RNA (crRNA). Its crRNA guides the Cas13a to an ssRNA target and then cuts other ssRNA molecules non-discriminately.

The alternative repair mechanism is called homology-directed repair (HDR) [51]. This is a more accurate DNA repair pathway that needs a homologous template to direct repair (Figure 3). Although less prone to introducing errors, HDR is commonly thought to be less effective than NHEJ and only happens at definite stages of the cell cycle. By establishing a homologous template that includes the desired DNA change, the HDR-based CRISPR technique is an excellent selection for knock-in (KI) experiments in diving cells.

Though advances in nuclease and delivery routes have made CRISPR more efficient, low repair efficacies and the production of indels at the target genomic site can be detrimental when constructing GE therapies for clinical use. Furthermore, the application of Streptococcus pyogenes Cas9 (SpCas9) in recent research has revealed additional drawbacks, for example, its high mutagenic activity, strict PAM requirement, and large size [53]. CRISPR/Cas9 can only target locations with PAMs with the NGG sequence, which restricts GE at target sites with G-rich sequences.

The Cas12a (formerly known as Cpf1) was discovered in the bacterium Francisella novicida [54]. Cas12a fixes this problem in the interim by detecting the T-rich target sites, relying on a T-rich PAM, and causing a staggered nick in double-stranded DNA. The CRISPR system utilizes Cas12a in a distinct way from Cas9. Contrary to the Cas9 system, which needs an RNA composed of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), the Cas12a system requires only crRNA (Figure 3). Thus, a single RNA molecule would be sufficient to continue the process, since in this case, Cas12a leaves behind a sticky end rather than a blunt one after cleavage (Figure 3).

Consequently, genes of desire can be consciously placed into vectors using CRISPR/Cas12a technology. As a result, Cas12a is more efficient than Cas9 [55]. The Cas12a family proteins are a class of Cas9 orthologues, and the proteins’ HNH endonuclease domains are structurally different from Cas9’s [56]. The HNH endonuclease domains, which stand for the three most conserved amino acid residues: two Histidines (H) and one Asparagine (N), play critical roles in binding a catalytic metal ion, DNA cleavage, particularly in genome conservation, host defense, and the movement of genetic elements [51]. In practice, the discovery of Cas12a offered a clearer, more limited substitute for the CRISPR toolbox, opening the possibility of more precise genome editing. Another benefit of Cas12a over Cas9 is its ability to target a broader range of genomic sites. Recently, researchers created Cas12a proteins with uridine-rich 3’ ends, in addition to a complementary 20 bp target site, to increase the efficacy of Cas12a in inducing indel mutations at target sites.

In 2016, a new RNA-directed RNA endonuclease, Cas13a (C2c2), was described from the bacterium Leptotrichia shahii (LshCas13a) [57]. The Cas13a protein consists of the crRNA-recognition (REC) lobe and nuclease (NUC) domain [48]. The REC lobe has an N-terminal domain (NTD) and a domain called helix-1 or Helical-1. The NTD is a non-conserved region of Cas13a that is made of a larger subdomain containing an ordered portion consisting of seven α helices and a disordered portion, and a smaller subdomain having three α helices, a β-hairpin, and a β-sheet [48]. Cas13a is guided by its crRNA to an ssRNA target and then cuts other ssRNA molecules non-discriminately. This cleavage pattern of Cas13a, called collateral cut, has been used to advance various diagnostic technologies. As shown in Figure 3 and Table 1, the CRISPR-Cas13 system comprises two components: the Cas13 effector protein and a crRNA of 64-66 nucleotides [58]. Thus, unlike the CRISPR/Cas9 and CRISPR/Cas12 systems, which target DNA, the CRISPR/Cas13 system targets RNA.

Following the earlier CRISPR/Cas GE technologies and the concerns about the genotoxicity of DSBs and the need to address HDR’s low efficacy, further development of “second-generation” CRISPR approaches has enhanced GE without depending on DSB generation or HDR. Continuing research in GE has led to the prompt iteration of CRISPR technologies, such as base and prime editors (BEs and PEs, respectively) (Figure 4), allowing precise nucleotide modifications without the requirement for generating harmful DSBs.

Figure 4 Basic overview of the base editing and prime editing systems. Base editors consist of a catalytically impaired Cas9, sgRNA, and a deaminase. Base editors mainly facilitate C-to-T or A-to-G conversions without forming DSBs. Prime editors comprise a Cas9 nickase, an engineered reverse transcriptase, and a pegRNA, which comprises a primer-binding site (PBS) and reverse transcription (RT) template encoding the desired edit. Prime editors can make small insertions and deletions and can ease all types of point mutations. The editing efficiency of each tool can be promoted through Artificial Intelligence (AI)-driven approaches. Modified from [59].

Base and primer editing is an elegant procedure for replacing single nucleotides without needing donor DNA templates or DSB formation [60]. The development of BEs and PE demonstrated the potential of new technologies to edit non-dividing cells, a major advance. It has solved the limitations of traditional GE technologies. By improving the structure of the deaminase, optimizing RNA design, and using ribonucleoprotein (RNP) methods to reduce off-target effects, they have improved efficiency and stability [42,61]. The advanced invention offers a much more tailored method to GE, with particular technologies specifically suited to certain types of edits or delivery modes. Two classes of BEs were advanced: Cytosine BEs (CBEs), which have catalytic domains originating from cytidine deaminases and an uracil glycosylase inhibitor (UGI) domain, mediate C-to-T conversion [62]. In turn, adenine BEs (ABEs) make A-to-G conversions utilizing an adenosine deaminase domain from the tRNA-specific deaminase TadA that has been obtained by directed evolution to act on ssDNA. Though BEs enable more precise control over editing outcomes, they do suffer from several drawbacks, including limited efficiency, substantial off-target activity, and genotoxic effects [63].

The application of the BE technique highlighted several limitations; only some types of base changes can be carried out. Particularly, transversion mutations (i.e., the conversion of a pyrimidine base to a purine base, or vice versa) are not achievable [62]. In addition, this technique cannot be used to generate indels, which can limit its functionality. Still, the impressive efficacy and ability to make multiple edits in one cell make BE an attractive tool for therapeutic GE.

In contrast to BE, a novel technique (PE) was developed to allow both base conversions (transition and transversion) [59,62]. Prime editing is a Cas9-based strategy developed to produce targeted point mutations, insertions, or deletions in an HDR-independent manner [59,60]. The PE consists of a prime editing guide RNA (pegRNA) and a fusion protein construct made of Cas9 nickase with deactivated HNH domain and an engineered reverse transcriptase (RT) domain [59,62]. The pegRNA includes a 3’-terminal sequence that is complementary to the nontarget strand (NTS) of the desired genomic target, and that carries the intended mutation(s). Cas9 creates a nick in the NTS, which then hybridizes with the complementary extension of the pegRNA [59,62]. To date, PE has been successfully used in different organisms and cell types. However, depending on the planned edit, target site sequence, and cell type, PE efficacies are greatly variable and often low. Novel PE variants with enhanced performance continue to be developed, including systems that enable the formation of more extensive edits using pegRNA pairs [64].

Prime editing works by inducing single-stranded breaks (SSBs) in the genome, which are then repaired error-free (Figure 4). This needs a fusion protein (a Cas9 nickase) combined with an RT enzyme that identifies the genomic target location and creates an SSB into the non-target strand. Next, the released 3’ DNA strand connects to the 3’ terminal zone of the pegRNA – containing the desired edit – and is reverse transcribed by the RT enzyme. Once the flap is cleaved, the DNA is then ligated, and the edited DNA has been effectively integrated into the genome.

The major benefit of the PE technique is that it avoids introducing DSBs while still delivering accurate DNA alterations, although the process’s efficacy still needs optimization [59,62]. In view of its diverse functionality, PE could theoretically correct up to 89% of common genetic variants associated with human disease [65]. Some PE-based methods have been advanced that use this technology to detect and cure diseases [66]. Although the method and early proof-of-perception studies seem promising, PE has yet to enter clinical trials.

A recently reported technological development in CRISPR GE that is similar to PE is called Programmable Addition via Site-specific Targeting Elements (PASTE) [62]. The similarity between PE and PASTE is the utilization of a CRISPR/Cas9 nickase. However, the difference is that Cas9 is the merger of two enzymes — RT and serine integrase, to enable the integration of large DNA fragments [59,62]. In addition, like PE, PASTE displays no off-target activity — so no unintended genomic alterations occur at locations other than the target site.

CRISPR’s Role in Diabetes Research. Gene regulation differs significantly between human islets and laboratory animal or islet-like cells derived from human cell lines [67]. For instance, genes encoding the transcription factors (Homeobox proteins SIX2 and SIX3) are expressed in adult human β-cells, but not in rodent β-cells [68,69], stressing the necessity for genetics in primary human islets. Discovering and editing genetic variants implicated in increased T2D risk is another application of CRISPR/Cas9. CRISPR-mediated GE or control of gene expression in primary human islet cells has not previously been reported. However, using CRISPR/Cas9 and CRISPR/dead or deactivated Cas9 (CRISPR/dCas9)-based enhancer activation (CRISPRa) demonstrated the feasibility of engineering coding and non-coding regulatory genomic loci in primary human islets [1]. These studies disclosed a vital function of the transcription factor (Pancreatic and Duodenal Homeobox 1; PDX1) in primary mature human islet β-cells, as expected from mouse and human genetics, and identified functional target genes of regulatory elements that harbor T2D-associated variants. CRISPR/Cas9 can also be used to decrease the expression of genes linked to glucose dysregulation and insulin resistance, offering a targeted approach to diabetes prevention [14]. Thus, it is possible to expand the experimental range for dissecting genetic mechanisms causing human diabetes.

CRISPR screens have been applied primarily in basic cell biology, cancer biology, immunology, and virology; however, they have rarely been used in diabetes research [70]. A possible reason is that diabetes-associated research can be more complex, often involving cross-talk between organs or cell types, making it challenging to apply a CRISPR screen. CRISPR’s tools have opened a world of possibilities in the quest to comprehend and eventually treat diabetes. In diabetes research, the CRISPR/Cas9 technique has been applied across various fronts, primarily for precise GE applications. Such examples involve the perturbation and study of T2D risk genes in vitro [71], the creation of genetically edited brown-like adipocytes to increase glucose tolerance and insulin sensitivity [72], and CRISPR-based editing of stem cells to modify disease-causing mutations and inhibit immune rejection in β-cell transplantation [73].

Applications of CRISPR/Cas9 in T1D research and therapy have increased rapidly. A genome-wide CRISPR knock-out (KO) screen was performed to identify genes that regulate pancreatic β-cell protection against autoimmune destruction [74]. The autoimmune killing provided a strong life-death selection pressure on transplanted pancreatic β-cells. Researchers [74,75] identified several gene mutations, including a candidate human T1D GWAS gene (RNLS) on the 10q23.31 risk locus. This gene encodes for the enzyme FAD-dependent amine oxidase (Renalase), which decreases intrinsic stress in β-cells and defends the cells from autoimmunity. CRISPR/Cas9 is being examined to modify the immune response associated with T1D. By editing genes implicated in immune regulation, researchers hunt for ways to suppress the autoimmune attack on β-cells, preventing or delaying disease development [14]. More recent reports [3] concentrate on using CRISPR/Cas9 to engineer autoreactive T cells, either by KO the T cell receptor genes or by KI protective genes to leave them less pathogenic. Another promising application includes β-cells to improve their continued existence and function post-transplantation. Scientists are also exploring the potential of CRISPR/Cas9 to modify genetic variants associated with high T1D risk using patient-derived induced Pluripotent Stem Cells (iPSCs) [76]. Moreover, genome editing is being used to produce more advanced animal models of T1D, enabling better identification of disease mechanisms and assessment of potential therapies [3,24].

CRISPR-Based Treatment.

(i) Manipulating Insulin Response. Controlling insulin response using CRISPR-based techniques has been indicated as a procedure that will dramatically shift traditional treatments of DM. This area emphasizes the perception of how genes affect insulin secretion and its sensitivity. By effectively modifying these genes, scientists hope to improve insulin signaling in cells, particularly in individuals with T2D, where insulin resistance is predominant. Because this approach is relatively new, evidence of its efficacy in human models is still needed. For instance, CRISPR systems have been implemented to drive differentiation, transdifferentiation, and reprogramming of different mouse and human cell types [77].

Remarkably, the diversity of genes examined using the CRISPR procedure to target DM spans those involved in obesity to those involved in DM itself, providing ample room for testing in animal models. Recently, significant progress in gene therapy has been achieved, providing promising solutions for the challenges in T2D therapy, opening new paths for personalized and effective therapeutic strategies. The CRISPR-based technology is robust and capable of diverse applications, including correcting specific mutations associated with T1D and promoting insulin production in T2D. Utilizing the CRISPR/dCas9 system to reprogram the expression of the gene Thioredoxin-interacting protein (TXNIP), which is involved in T2D pathogenesis, could offer therapeutic benefits, particularly in protecting pancreatic β-cells, promoting insulin sensitivity, and mitigating inflammation [25,78].

(ii) Manipulating β-Cell Function. Controlling the function of β-cells in the pancreas represents an exciting frontier in diabetes investigations. These cells produce insulin, and their failure causes diabetes. This factor positions CRISPR at the forefront of therapeutic strategies to cure diabetes. Therefore, promoting the proliferation or function of β-cells through CRISPR could theoretically lead to restoration of insulin production. Cell-mediated therapy for T1D follows a stepwise differentiation procedure that transforms iPSCs into mature β-cells. In this process, cells progress through successive stages including definitive endoderm, primitive gut tube, posterior foregut, pancreatic progenitors, endocrine precursors, and immature β-cells [3]. The obtained stem cell-derived β-cells (SC-β-cells) can be encapsulated and co-transplanted into T1D patients.

CRISPR/Cas9 has demonstrated efficiency in using ex vivo GE in embryonic stem cells and the human iPSCs (hiPSCs) obtained from the patient [4,79]. These cells are transformed into perfected pancreatic βcells. In this procedure, various methods are used, including cell-based treatments (e.g., stem cells and brown adipocytes) and targeting specific genes related to diabetes.

The invention of an autonomous closed-loop system that reproduces pancreatic β-cell function or biologically controlled insulin secretion, for example, islet transplantation, could be a revolutionary option. A recent study [80] reported that a T1D patient began producing insulin after receiving β cells derived from stem cells. Similarly, a T2D patient in Shanghai stopped using insulin following undergoing an islet cell transplant [81]. These cases illustrated the promising possibilities of stem cell therapies to exclude the dependence on insulin and decrease the need for donor organs and immunosuppressive drugs [81].

The glucose supervising and insulin dosing behavior derived from integrated continuous glucose monitoring (CGM) and connected insulin pens (CIPs) data aid in managing DM and can be used to reach optimal glycemic control [82]. The unique feature of this method is the ability to initiate a self-sustaining cycle where the body’s insulin production is improved, not only temporarily fixed.

Nevertheless, although pancreatic islet or β-cell transplantation provides a potential cure, it’s important to tread with care; while aims are noble, disruptions of β-cell function may result in an imbalanced insulin response, leading to hypoglycemia, which is no easy matter. Furthermore, it encounters problems such as restricted donor availability and poor cell engraftment. In addition, this procedure carries side effects associated with the infusion procedure itself and the need for lifelong immunosuppression. More importantly, the transplantation includes infusing cells into the liver’s portal vein, usually under local anesthesia. Potential short-term complications include: bleeding at the infusion site, which may necessitate a blood transfusion, as well as thrombosis in the portal vein, which may lead to graft loss.

(iii) Manipulating Animal Models. CRISPR/Cas9 allows the creation of novel genetically modified animal models for diabetes and evaluates the effectiveness of various therapeutic strategies in treating the situation [4,79]. Studies in mice have confirmed that the diabetic condition can be reversed by specific gene targeting. Signal transducer and activator of transcription (STAT) has been shown to mediate the activation of Neurogenin-3 (NGN3) in acinar cells, reprogramming them into β-cells in diabetic mouse models [83]. The examination of the activating mutation, STAT3K392R, on pancreatic development utilizing iPSCs originated from a patient with neonatal diabetes and pancreatic hypoplasia indicated that this mutation initiated premature endocrine differentiation by direct induction of Neurogenin 3 (NEUROG3) expression. Luckily, the disease phenotype was totally reversed using the CRISPR/Cas9 technique to correct the STAT3 mutation [79].

Researchers [84] have combined the CRISPR/Cas9 GE method with the iPSC derived from the skin of a diabetic patient with a rare genetic type of insulin-dependent diabetes called Wolfram syndrome. Patients with the syndrome get diabetes in childhood or adolescence and immediately need insulin-replacement therapy, requiring insulin shots multiple times every day. In many cases, the syndrome may cause an early death. In their study, they first obtained skin cells from three patients and converted the cells into iPSCs utilizing a mix of growth factors and nutrients. They transformed stem cells into insulin-producing cells, then applied CRISPR/Cas9 GE to correct a single-nucleotide mutation in the Wolfram Syndrome 1 (WFS1) gene that causes diabetes, producing three clonal iPSC stem cell lines. CRISPR/Cas9 genetically engineered patient-derived β-cells were implanted into mice to reverse diabetes in the animals. These results indicated that the CRISPR/Cas9 strategy may hold promise as a treatment for diabetes, mainly the forms produced by a single gene mutation. In the future, this approach may be suitable for some patients with the more frequent forms of diabetes, such as T1D and T2D.

3.2 Present Research Outcomes

3.2.1 Pioneering Work in Animal Models

Success stories in animal models for improving insulin sensitivity highlight the substantial progress being made with CRISPR in diabetes research. Researchers have reported cases in which diabetic mice showed marked improvements after GE. For instance, scientists have successfully targeted genes involved in insulin production, achieving promising results that could lead to potent therapies for humans in the future. Four distinct sgRNAs targeting about 50 to 150 base pairs (bp) upstream of the human Uncoupling protein (UCP1) gene have been engineered with a synergistic activation mediator (SAM) to enhance CRISPR activity [72]. The UCP1 gene encodes a mitochondrial carrier protein that produces brown and beige tissue of mammals. It is implicated in non-shivering adaptive thermogenesis, and its expression is associated with increased energy expenditure, making it a cautious candidate for anti-obesity treatment [77]. The engineered adipocytes inhibited diet-promoted obesity and improved metabolic syndrome in mice. The CRISPR/SAM model was used in the human white preadipocytes from two DM subjects. The results showed that UCP1 mRNA was amplified 6000-fold, accompanied by a 20-fold increase in UCP1 protein expression. The obese mice that developed human brown-like adipocytes showed sustained improvements in glucose tolerance and insulin sensitivity. Despite the study’s encouraging outcomes, the Wang team [72] acknowledged some drawbacks and noted that further investigation is required to establish human applicability.

Another pioneering work [85] developed an sgRNA targeting the gene encoding fatty acid-binding protein 4 (Fabp4) in white adipocytes. This targeting was based on the finding that silencing the Fabp4 gene was an effective approach to stimulate weight loss and metabolic recovery in high-fat diet-induced mice. It has been reported [77] that targeted delivery of the CRISPR system against Fabp4 in white adipocytes decreases the expression of this protein. The latter protocol led to reduced body weight and decreased inflammation with the recovery of hepatic steatosis in obese mice. Additionally, insulin sensitivity was substantially improved in the experimental mice. It was concluded that this approach offered a simple and safe strategy to treat obesity and its complications while concurrently improving several metabolic variables like insulin sensitivity [85].

While rodent models are valuable for studying diabetes, the usage of animal models metabolically closer to humans (pigs or non-human primates) is a more efficient way to understand the pathogenesis of human diseases using the CRISPR/Cas9 system [86,87]. Although diabetes investigations can benefit from using pigs as a model due to their shared physiology and metabolic pathways with humans, the lack of pig models exhibiting diabetes symptoms represents an obvious handicap. For this purpose, the INS gene is first altered in somatic cells using the CRISPR/Cas9 system [88]. Next, somatic cell nuclear transfer carrying the adjusted INS gene caused the production of pig embryos with an INS KO phenotype. Insulin KO piglets exhibited hyperglycemia and glucosuria, confirming the effectiveness of CRISPR/Cas9-based generation of new pig models, which could be more appropriate for drug development and islet transplantation research compared to rodents. Though experimental animal models are required to replicate human diseases, genetic and metabolic differences between species can occasionally prevent them from adequately mimicking human disease, hindering the development of an effective treatment. Thus, though these success examples are morale promoters, there remains the important hurdle of translating these outcomes into human trials, making this a double-edged sword.

3.2.2 Success Stories in Cells

Diabetes involves multiple organs and cell types interacting with one another. But it is still possible to use one cell type to investigate specific aspects of diabetes, which may cast light on comprehending diabetes on a systematic level. Here, a few such examples are documented. Managing β-cell function represents an exciting frontier in diabetes research. Beta cells in the pancreas secrete insulin, and when they stop working, diabetes ensues. So, augmenting the proliferation or function of these cells by CRISPR could hypothetically lead to the restoration of insulin-making. This factor places CRISPR at the forefront of treatment strategies aimed at curing diabetes. The CRISPR/Cas9 system provides a suitable method for producing pancreatic β-cells devoid of INS synthesis [79]. At the DNA level, the INS gene can be silenced with the help of the CRISPR/Cas9 by means of particular sgRNAs targeting the INS gene. As these cells will have all the characteristics of the β-cell (except for insulin synthesis), they will be an ideal model for ex vivo and in vivo assays of the efficiency of INS gene delivery vectors. To build this model, pancreatic β-cells are transduced with a silencing plasmid encoding the CRISPR/Cas9 protein, specific sgRNA, and an HR plasmid having the homologous regions to the INS gene [79]. However, it is imperative to tread cautiously; while goals are noble, disruptions of β-cell function may result in an imbalanced insulin response, causing hypoglycemia, which is no minor matter.

Because isolated human islets are a rare and precious resource for DM studies, human Pluripotent Stem Cells (hPSCs) represent a valuable alternative to pancreatic islet donors. iPSCs can be created from adult somatic cells by direct reprogramming and differentiated into β-like insulin-making cells [89]. Applying iPSCs offers a motivating alternative for DM therapy. A stepwise protocol was designed [90] to obtain iPSCs from human pancreatic cells using the CRISPR-Cas9 technique. Various studies have confirmed that the CRISPR approach can significantly reduce the cost and time required for chimeric organ production. But immune tolerance must develop before the technique can be applied in humans [91]. In comparison, the human embryonic S7 cells were verified to substantially outperform the iPSCs taken from pancreatic precursors in several aspects [92]. First, the S7 cells showed suitable glucose responsiveness and, at the same time, reversed DM four times more rapidly than iPSCs. Second, iPSCs produced fewer insulin-producing cells than their human embryonic stem cell counterparts. More research is needed to optimize the feasibility of the CRISPR protocol in cell therapy.

An effective GE program, inducible CRISPR (iCRISPR), a rapid platform for GE of hPSCs, was developed using TALENs and the CRISPR/Cas system [93]. This GE system enables scientists to control when and where the CRISPR/Cas9 apparatus is active, classically by using a chemical trigger (such as doxycycline) to switch it on or off. Eight of the transcription factors (pancreatic duodenal homeobox 1 [PDX1], Regulatory Factor X6 [RFX6], Pancreas Associated Transcription Factor 1a [PTF1A], GLIS3, Motor Neuron and Pancreas Homeobox 1 [MNX1], NEUROG3, Hes Family BHLH Transcription Factor 1 [HES1], and Aristaless Related Homeobox [ARX]) that are efficient in β-cell development in Human embryonic stem cells (hESCs) have been silenced using CRISPR/Cas9 and TALENs [94]. From a different perspective, a CRISPR/Cas9 model has been devised [1] to target the gene encoding PDX1, a crucial transcription factor for β-cell function that is altered in human diabetes. Indels were identified in about 66% of sequences by polymerase chain reaction (PCR). This method reported a meaningful decline in PDX1 protein expression in cells treated with the CRISPR system, suggesting specific targeting of PDX1 was feasible. It was reported that PDX1 loss impaired the physiological function of β-cells. Similar outcomes were obtained when targeting the potassium inwardly rectifying channel subfamily J member 11 (KCNJ11) gene, which led to β-cell loss of function [1]. Overall, the researchers concluded that CRISPR/Cas9 was an efficient strategy to target any gene of concern in human islet cells [1]. More recently, in 2024 [55], scientists have explored the utilization of adeno-associated virus (AAV) vectors to carry the genes PDX1, NGN3, and MAFA to promote β-cell neogenesis.

The major problem with applying Cas9 or any other Cas nuclease to edit iPSCs is the elevated sensitivity of iPSCs to DSBs [95] via p53 activation [96]. Methods with transient p53 inhibition resulted in the genomic instability of the generated edited iPSC clones [97]. Consequently, safer and more precise genomic editors should be applied for therapeutic iPSC editing.

3.2.3 Preclinical and Clinical Trials: Overview

Advancements in porcine islet transplantation have been considerable in recent years, with some preclinical studies indicating long-term glycemic control in non-human primates. Progress in genetic engineering has enabled the production of pigs with several genetic modifications, focusing on issues like hyperacute rejection and coagulation incompatibilities [98]. These “humanized” pig islets confirm developed compatibility with the human immune system and improved functionality. Recent research also addresses optimizing separation and culture protocols to amplify the yield and quality of porcine islets [99].

In people, the impact of clinical trials using CRISPR implies a cautious step toward studying GE in the treatment of diabetic individuals. These trials are critical as they help to verify the efficacy and safety of different medical interventions for the disease in human populations. Scientists have started testing CRISPR strategies to modify genes linked to insulin regulation in small-scale cohorts of participants with various types of diabetes. While the promise is considerable, the regulatory basis governing these trials demands reliable adherence to ethical morals and robust monitoring conditions. One prominent feature of these trials is their well-defined design, offering an obvious means to gauge CRISPR’s viability. However, preserving participant safety remains vital, as any unanticipated side effects could stall the field’s momentum, underscoring the need for longer-term studies to fully gauge efficacy and safety.

A clinical study was conducted to assess the efficiency of a stem cell therapy, VCTX210, in constructing insulin for T1D patients. The VCTX210 is a combination product that consists of allogeneic pancreatic endoderm cells (PEC210A), which have been genetically engineered using the CRISPR/Cas9 technique to increase immune evasion and survival, and a punctured device that transports and retains PEC210A cells. Stem cell-derived islets from hPSCs, as a feasible source for T1D treatment, show promise in clinical trials [3] but remain hampered by functional immaturity, transcriptional identity issues, and the inability to control the ratios of β, α, and δ cells. Throughout in vitro differentiation, a large number of cells might acquire an undesired identity, complicating their application in T1D therapy. Although in vivo transplantation can improve the function of these cells, boosting the in vitro generation processes and certifying safety, particularly regarding unattached cell types that could develop into tumors, and guaranteeing genetic stability, remains critical. Advances in scalable production, characterization protocols, and cryopreservation will be crucial for the clinical implementation and availability of these therapies.

3.3 Limitations of Using CRISPR for Diabetes

Although the CRISPR system holds enormous promise and offers a novel approach to the therapeutic strategy of DM at the genetic level, the likelihood of complete revolutionary clinical success is slim. Challenges that surround CRISPR-driven treatments for DM can be grouped into: (a) technical limitations, (b) approval and monitoring processes, and (c) safety and ethical considerations. These issues have impeded the applicability of CRISPR in cell engineering [13,100,101] and thus necessitate rigorous assessments and optimization of delivery methods. Researchers are actively working to refine CRISPR techniques to reduce these risks, enabling safer and more efficient applications in DM treatment and modeling and paving the way for novel therapeutic strategies.

3.3.1 Technical Limitations

Off-Target Effects. Off-target effects occur when the CRISPR procedure inadvertently changes the genome at unintended locations, such as chromosomal translocations, deletions, or inversions, or induces DNA damage and stress response pathways [60,101]. Off-target editing remains a major concern in clinical settings for CRISPR-based therapies, as these effects pose crucial hurdles to the implementation of CRISPR systems [47]. This is vital because it can lead to unexpected consequences, including carcinogenesis, potentially establishing new health issues rather than solving prevailing ones. For example, editing a gene intended to improve insulin regulation could unintentionally KO or alter neighboring genes, leading to undesired results. While adjusting insulin response could be revolutionary, it poses risks of off-target effects, where accidental edits could abruptly worsen health concerns, underlining the need for meticulous research and confirmation. The key feature of off-target effects is their unpredictability. They are often difficult to detect until after the fact, making surveillance challenging. Enhanced major efforts to advance robust and sensitive approaches for the prediction and detection of off-target edits and to increase the specificity of CRISPR genome editors have been reported [102].

To minimize these off-target mutations, researchers at Osaka University have developed a new, safer, and more precise alternative to CRISPR/Cas9 for the GE technique called Nicking-Induced Co-conversion with Error-Prone DNA Repair (NICER), which causes significantly fewer off-target mutations than CRISPR/Cas9 editing [103]. NICER was capable of restoring the expression of disease-causing genes in cells derived from genetic diseases, implicating compound heterozygous mutations. While some researchers aim to minimize off-target effects through advanced targeting techniques, the risk remains a key consideration when evaluating CRISPR’s utility in treating diabetes. Since the NICER technique does not involve DSBs or exogenous DNA, it appears to be a safe alternative to traditional CRISPR/Cas9 technologies.

Rapid advances in Artificial Intelligence (AI) can provide high-level solutions to these off-target problems. By leveraging large datasets from diverse experiments, AI enhances gRNA design, predicts off-target activity, and improves editing efficiency [104].

Delivery to Target Cells. The therapeutic efficacy of CRISPR systems is directly shaped by the act of the delivery system [47]. This is critical, as inefficient delivery routes may lead to suboptimal outcomes or failure. To ensure the efficient transfer of CRISPR/Cas9 components to target tissues and cells, scientists have explored various delivery methods. Delivery methods for CRISPR to targeted tissues and cells have been explored and tested, including viral and non-viral vectors, as well as more innovative approaches, each with distinct benefits and shortcomings. A major technical bottleneck for most in vivo and ex vivo GE applications remains the size and the efficient delivery of the editing effectors, to expand the range β of cell types and tissues that can be targeted. Even the BEs, PEs, and their alternates are much larger than conventional CRISPR systems, which challenges recombinant protein generation and delivery as RNP cargo [60]. Various approaches have been investigated to reduce the size of BEs and consequently improve their clinical applications [105]. These strategies include trimming nonessential domains and substituting large components with smaller substitutes without compromising the editing efficiency.

The delivery of CRISPR elements is limited by specific restrictions of the delivery vectors and target cells or organisms. Developing efficient, safe delivery of CRISPR-based therapies to target cells demands special attention before translating CRISPR-based therapies from the laboratory to clinical applications [14,46]. In CRISPR/Cas9-based treatments, the selection of an effective delivery mechanism is challenging and time-consuming since the CRISPR/Cas system is complex. This is because components must be transported to the nucleus to exert their influence on the nuclear genome, overcoming tissue and cell membrane barriers [106].

Viral delivery is the major choice for gene therapy; however, it poses serious safety issues like the possibility of adverse immunogenicity [107]. For this reason, nanoparticles (NPs) have been recommended to address the challenge of targeted delivery in genome editing. The unique feature of using NPs for CRISPR transfer lies in their ability to avoid immune detection; their efficiency in human applications continues to be under investigation. Thus, unresolved concerns about delivery systems can influence CRISPR’s performance in treating diabetes, underscoring the need for ongoing research in this domain.

T2D could be treated by a low-risk treatment approach, such as a CRISPR/Cas9-based technique that can effectively downregulate the DPP-4 enzyme. Cas9-RNP complex can be constructed before transport into cells as a recombinant nuclease (Cas9) and an RNP. This Cas9-sgRNA multiplex is planned to alter the DPP-4 gene. A lecithin-based liposomal nanocarrier particle (NL) was generated to deliver the Cas9-RNP complex [88]. A cationic polymer was incorporated with the Cas9-RNP complex to construct a negatively charged lipid construction of the NL and boost encapsulation efficacy. This is so since electrostatic interactions are a major determinant of loading efficiency [108]. Because the liver normally metabolizes lecithin, NLs are also well-suited for addressing liver diseases from a biodistribution perspective.

Negatively charged lipids and charge-compensated multiplexes naturally interacted electrostatically, forming NL spheres with a similar size distribution to self-assemble. The use of a nanocarrier and the CRISPR/Cas array reduces the risk of hypoglycemia and enhances patient compliance while mimicking endogenous insulin secretion via external stimulation. The genome platform is ideal for treating genetic and chronic human disorders, as it offers excellent biocompatibility, reduced cytotoxicity, and high solution stability, unlike unprotected protein therapies, which suffer from reduced delivery efficacy due to enzymatic degradation.

Polymer-based NPs have been used to deliver CRISPR components and other silencing strategies, targeting genes such as Bone Morphogenetic Protein 9 (BMP9) and Phosphoenolpyruvate carboxykinase (PEPCK), therby efficiently decreasing glucose levels and enhancing glucose tolerance while lowering inflammation and insulin resistance [16]. Although there has been significant progress in efforts to identify an effective approach to curing DM, more work is required to overcome the limitations inherent to this technique and achieve the goal of curing DM.

Approval and Monitoring. Gene-editing tools confront regulatory uncertainties, particularly regarding long-term safety measurements and ethical concerns [109]. The key feature of safety evaluations lies in their depth. These assessments typically require comprehensive preclinical and clinical trials to determine the short- and long-term effects of GE interventions. The approval process for CRISPR’s application to diabetes is lengthy and complex, involving multiple regulatory bodies assessing the technology’s safety and efficacy before human use. This long process allows the identification of likely adverse reactions, ensuring that CRISPR therapies are truly more beneficial than harmful. Though it may look meticulous, such assessments are crucial, as handling genetic material can have significant effects on human health. This aspect is crucial to achieving the overall goal of patient safety and maintaining public trust in medical innovations. Once therapies are in practice, it becomes important to track their effects over time to confirm they deliver anticipated advantages without severe side effects.

The significance of long-lasting post-marketing surveillance cannot be overemphasized; without it, the dangers associated with unpredicted outcomes could remain unseen for years [110]. A key aspect of long-term monitoring is patient follow-up and data collection to ensure the long-term effectiveness of CRISPR-based interventions. Although this essential feature may at first appear burdensome, it significantly contributes to a broader understanding of genetic therapies. The promise of benefits includes not only well-established safety data but also useful awareness that can further boost future CRISPR applications.

Safety Assessments and Ethical Considerations. Added to the technological obstacles, CRISPR techniques, like traditional GE and therapy, still raise major concerns about immunogenic toxicity. This response develops from the usually used Cas9 proteins from S. pyogenes and S. aureus, which trigger an immune reaction in humans [111]. The introduction of CRISPR inhibitors offered a solution to immunogenicity by inactivating the GE enzymes after DNA cleavage [112]. Even the modified Cas9 has to be carried by a vector designed to escape triggering the host immune response [46]. To reduce the possible immunologic reaction against Cas9 by pre-existing antibodies in human serum, the use of new Cas enzymes like the Cas12e and Cas12d from soil bacteria has been reported [46]. The use of plant-derived exosome-like NPs as a delivery system of CRISPR/Cas9-based therapeutics has been welcomed for safety goals due to the wide differences between plant and mammalian pathogens [113].

As for ethical considerations, they play a key role when debating CRISPR technology. The significance of CRISPR in studies of diabetes lies not only in its application but also in the ethical debates and societal concerns it raises, which warrant attention [109]. This component covers a broad range of concerns, beginning with the implications of genetic reformation to issues of consent and the possible misuse of technology. One main ethical fear is whether modifying genetic material without a full understanding could result in unforeseen consequences for patients and their descendants.

The distinguishing quality of ethical concerns lies in their multifaceted nature. While GE holds significant potential for treating conditions like diabetes, it raises queries regarding the fairness of access to such therapies. In addition, the possibility of developments beyond therapeutic events could lead to societal divides. These ethical implications are not just marginal; they are integral to defining how CRISPR can be responsibly applied and accepted in medical practice. Patients must be assured that their data will be used ethically and protected from misuse. So, we need to conduct additional studies on CRISPR-Cas9 and apply it in appropriate contexts to support its future development. The nation and the government must allocate sufficient funds for a comprehensive examination of this technology.

Affordability and Accessibility. One of the leading challenges in diabetes concerns is the economic burden of long-term administration [10]. Despite it being on the WHO’s list of essential medicines, the cost of insulin remains a significant barrier, particularly for T1D patients. Furthermore, novel pharmacotherapies (e.g., SGLT2 inhibitors and GLP-1 receptor agonists) remain inaccessible to people in low- and middle-income countries due to high costs and the absence of healthcare infrastructure [114].

3.3.2 Limitations of This Study

Although this article provides an overview of research on cell-CRISPR-based technologies for DM, some limitations remain. Firstly, it is hard to include details about the delivery strategies of the CRISPR/Cas system and the influence on the selection of the CRISPR technique. Secondly, some literature may have been excluded due to bias in the database or language, such as publications from Cochrane and Embase, as well as non-English-language publications. Moreover, the most innovative results, presented as posters or in conference proceedings, may have been skipped. Thirdly, the most recent, high-quality papers may not be included due to their weak citation counts, leading to a gap between the bibliometric assessment and the genuine world. Therefore, it is suggested that researchers focus on the most recent publications, particularly those in languages other than English.

4. Conclusions and Future Prospects of CRISPR in Diabetes Treatment

This review addressed recent advances in diabetes management, bringing together conventional and emerging methods, focusing on the revolutionary potential of cell- and CRISPR-mediated GE technologies. The future of GE technology in diabetes therapy is full of promise. Researchers can use CRISPR to cross-reference gene sequences from non-diabetic populations with those from diabetic populations. This may lead to the development of tests that accurately predict diabetes risk. Yet, this does come with its cautions since not every genetic marker applies to all individuals. With personalized medicine, treatment can be tailored to an individual’s genetic makeup. This characteristic allows more precise targeting of gene modifications relevant to their specific type of diabetes. However, this issue must be carried out cautiously to ensure that personalization does not inadvertently create new inequalities. Moreover, there’s a fine line between precise genetic profiling and unwarranted panic about possible health issues that may never manifest. The transformative power of CRISPR technologies holds genuine promise for cures and novel therapeutic strategies for DM life-long pathology of T2D. Longer-term studies are crucial to approve a drug’s safety because initial, shorter-term clinical trials are deliberately designed to determine efficacy and immediate safety in a limited, controlled population. They often cannot detect risks that only arise after prolonged exposure, such as late side effects, rare unfavorable events, or effects on a chronic condition.

Transplanting β-cells into severe diabetic mice quickly reverses their diabetes, offering a robust, functional cure. For the first time, researchers can use GE to correct monogenic forms of diabetes in stem cells derived from patients’ own cells. They took patient skin cells, converted them into stem cells, used CRISPR/Cas9 GE to correct a diabetes-causing mutation, and then transformed the edited stem cells into the insulin-secreting β-cells the patient lacks. It is predicted that CRISPR/Cas techniques have the potential to offer long-lasting, highly beneficial, cost-effective, and side-effect-free personalized treatments for the incoming adverse event that is haunting mankind. However, research on the topic appears cheerful only because of the scarcity of data and needs more effort to support long-term animal trials on the issue.

The future of CRISPR holds great promise, yet advancing its application in diabetes treatment demands ongoing studies and optimization to overcome complex hurdles with diligence and care. Gene editing remains one of the most magnificent advancements in molecular biology and holds promise for treating diabetes. Besides the technical challenges, international organizations hold a responsibility to develop financially sustainable models of strategic investments in medicine to ensure that benefits cover most patients and address the most urgent clinical needs. The present review can be further expanded to support in-depth research on this topic and to enable more discoveries in clinical GE technology. The path is long, and much effort is needed to realize the promises of CRISPR. The ultimate goal after using CRISPR is to achieve a substantial reduction of diabetes complications. Applying CRISPR alongside conventional medications and lifestyle changes may significantly improve the chances of more effective diabetes management. The profits could be life-changing, yet the way to common application stands tense with both hope and challenge. Given the early stage of research, the outcomes may raise some unknowns. Establishing GE associations and research networks devoted to diabetes permits coordinated efforts to advance CRISPR/Cas9 applications. It is essential for prospective diabetes therapeutic strategies to take these long-term consequences into account. This review article did not focus on analyzing productive countries or the corresponding author’s country for different aspects of DM. Future literature reviews may focus on bibliometric analyses of detection, classification, and various approaches to diabetic treatment.

Abbreviations

Acknowledgments

The author apologizes to those colleagues whose work is not cited due to restrictions on the number of references.

Author Contributions

The author confirms that he was solely responsible for the conception, design, analysis, interpretation, drafting, and final approval of the article.

Funding

This work received no funding from internal or external sources.

Competing Interests

The author declares no conflict of interest.

Data Availability Statement

No datasets were produced or analyzed during the current study.