Advances in epigenetic editing technology have paved the way for novel therapeutic approaches in various diseases, including diabetes. One promising approach is the CRISPR-dCas9-mediated editing of CpG islands, which could potentially target gene expression through epigenetic modifications. This review explores the role of CpG islands, their involvement in gene regulation, and how CRISPR-dCas9 can be used to edit these regions for potential diabetes treatment, focusing specifically on the TXNIP gene.
CpG islands are short stretches of DNA that are densely packed with cytosine-guanine (CpG) dinucleotides. They are commonly found near gene promoters, playing a critical role in gene regulation due to their influence on transcriptional activity (Bird et al., 1985; Bird, 1986). The unique feature of CpG islands is their high CpG density, their largely unmethylated state, and their strong association with active gene promoters (Gardiner-Garden and Frommer, 1987). Methylation of CpG islands, however, can lead to transcriptional silencing, making these regions key targets in epigenetic regulation.
Several computational tools have been developed to identify CpG islands within genomic sequences, including CpG Island Searcher, IslandPicker, and PromoterScan (Deaton and Bird, 2011). These tools use algorithms that search for high CpG density, appropriate sequence length, high GC content, and an observed/expected CpG ratio greater than a specific threshold (Choy et al., 2010). These features are essential for accurate identification and analysis of CpG islands, particularly in their role in regulating gene expression.
DNA methylation, the addition of methyl groups to the cytosine base in CpG dinucleotides, is a powerful epigenetic modification that silences gene expression when it occurs in gene promoters (Lee and Lee, 2012). Methylation interferes with transcription factor binding and recruits proteins that compact chromatin, thus reducing gene expression. Conversely, unmethylated CpG islands are typically associated with actively transcribed genes (Lee et al., 2015; Yoo et al., 2021). Factors influencing CpG island methylation include DNA methyltransferases (DNMTs), Ten-Eleven Translocation (TET) enzymes, chromatin accessibility, and environmental conditions.
In Dutta et al. (2005), identified a CpG island within the TXNIP gene's promoter region. This CpG Island, rich in cytosine and guanine dinucleotides, is a common target for epigenetic modifications, particularly DNA methylation. DNA methylation, the addition of a methyl group to cytosine residues in CpG dinucleotides, typically silences gene expression by hindering transcription factor binding or promoting chromatin compaction. Aberrant methylation patterns are implicated in various diseases, including diabetes.
Dutta et al. (2005) pioneered the discovery that hypermethylation of this CpG island correlates with decreased TXNIP expression in kidney cancers. Conversely, under normal conditions, the CpG Island is typically hypomethylated, leading to increased TXNIP levels. This dynamic regulation is essential for maintaining balanced cellular proliferation in both normal and cancerous kidney tissues (Dutta et al., 2005; Kim et al., 2021; Zhang et al., 2017).
A similar methylation pattern is observed in diabetic conditions. Hypomethylation of TXNIP correlates with elevated expression and disrupted glucose homeostasis (Zhang et al., 2017; Kim et al., 2021).
The methylation status of the cg19693031 site within the TXNIP gene has been linked to fasting blood glucose regulation in non-diabetic Taiwanese adults (Tsai et al., 2022). The relationship between TXNIP-cg19693031 DNA methylation (DNAm) and type 2 diabetes (T2D) is well-established, with strong correlations to HbA1c, insulin, and fasting glucose levels (Tsai et al., 2022). Hypomethylation at TXNIP-cg19693031 has been robustly associated with T2D, as well as elevated inflammatory biomarkers including VCAM-1, ICAM-1, MMP-2, sRAGE, and P-selectin. Notably, the connection between TXNIP-cg19693031 methylation and T2D persists independently of these inflammatory biomarkers (Xiang et al., 2021).
Further studies have also demonstrated a marked decrease in methylation across five TXNIP loci in individuals with T2D compared to healthy controls, where increasing methylation levels correspond to a reduced T2D risk. Interactions among TXNIP methylation, obesity, and hypertriglyceridemia were identified as contributing factors to T2D onset (Zhang et al., 2020).
Recent research has expanded on these findings, investigating how TXNIP methylation influences T2D risk in detail (Wu et al., 2024; Maeda et al., 2024). Two pivotal studies illustrate these associations, Wu et al. (2024) and Maeda et al. (2024) provide complementary insights into the role of TXNIP DNA methylation in type 2 diabetes (T2D) risk and glycemic regulation. Wu et al. (2024) in a nested case-control study, reported that higher methylation levels at TXNIP CpG sites 2–5 were associated with a 61–87% reduction in T2D risk, highlighting the protective potential of hypermethylation in this region. Similarly, Maeda et al. (2024) in a longitudinal study, found that hypomethylation at cg19693031 was linked to greater increases in fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c) over four years, suggesting that hypomethylation may impair glucose regulation and serve as an early marker of diabetes risk. Together, these findings underscore the importance of TXNIP methylation in diabetes pathogenesis and risk prediction.
2. CRISPR-Cas technology for editing CpG methylation
The discovery of CRISPR-Cas9 system has opened the door and revolutionized genome editing (Doudna and Charpentier, 2014). The catalytically inactive form of dCas9 allows for precise epigenetic editing without altering DNA sequences. By fusing dCas9 to epigenetic effectors like methyltransferases or demethylases, scientists can manipulate CpG island methylation. This offers a potential therapeutic avenue for diseases such as diabetes, where aberrant CpG island methylation affects key genes like TXNIP.
Table 1. A timeline highlighting the key developments in the area of CRISPR-dCas9 Mediated CpG island methylation and demethylation.
3. Current research and future directions
Today, CRISPR/dCas9-based CpG island methylation and demethylation are being explored for treating diseases related to epigenetic dysregulation, such as cancer, neurological disorders, and metabolic diseases. Challenges remain in optimizing delivery, minimizing off-target effects, and achieving long-term, stable modifications (Cano-Rodriguez and Rots, 2016).
3.1 In Vivo examples of CRISPR-dCas9 mediated epigenetic editing
CRISPR/dCas9-mediated CpG island methylation and demethylation in vivo have shown potential in animal models for understanding gene regulation and exploring therapeutic applications. Some prominent examples are presented in Table 2.
Table 2. Key in vivo applications of CRISPR-dCas9-mediated epigenetic editing.
4. CRISPR-dCas9-mediated reprogramming of TXNIP expression.
The CRISPR-dCas9 system can be leveraged to reprogram TXNIP expression by modifying its CpG island methylation status. For instance, CRISPR-dCas9-KRAB has been used to downregulate TXNIP by silencing its promoter region, reducing oxidative stress and improving glucose homeostasis. Alternatively, CRISPR-dCas9-TET1 can reprogram hypermethylated CpG islands in the TXNIP gene, leading to decreased TXNIP expression and improved insulin sensitivity.
5. Therapeutic potential of TXNIP reprogramming in diabetes
The ability to modulate TXNIP expression using CRISPR-dCas9 offers several therapeutic benefits for diabetes management. These include,
-Protecting pancreatic beta cells from apoptosis and oxidative stress.
-Improving insulin sensitivity in peripheral tissues.
-Enhancing glucose uptake and glycemic control.
-Reducing systemic inflammation associated with diabetes.
6. Conclusions
CRISPR-dCas9-mediated CpG island editing represents a novel and promising approach for diabetes treatment. By targeting key genes such as TXNIP, this technology has the potential to reverse hyperglycemia, enhance insulin sensitivity, and protect beta cells. While the potential is immense, further research is required to explore its therapeutic efficacy and address safety concerns.
Acknowledgements
The author would like to express his sincere gratitude to Professor Shinya Toyokuni, MD, PhD, Kyoto University (present location, Nagoya University), Japan, for supporting, guiding and supervising the discovery of Aberrant Methylation Mediated Regulation of TXNIP gene.
Ethical approval
No ethical approval is required for this study.
Declaration by authors
The authors' guidelines were used to generate the manuscript with the assistance of ChatGPT, an artificial intelligence program developed by OpenAI (which included the information mining, drafting and even for verification). However, the authors are responsible for the content and accuracy of the manuscript.
Data availability statement
The raw data are available in corresponding author and ready to submit when ask for it.
Informed consent statement
No informed consent was required to conduct the study.
Conflict of interest
The authors declare no conflict of interest.
Source of funding
This work received no funding from internal or external sources.
Author contributions
Khokon Kumar Dutta contributed to the design and writing of this review. The author critically reviewed the manuscript and agreed to submit final version of the article.
