Cosiquien Ronin Joshua S, Whalen Isaiah J, Wong Phillip, Sorensen Ryan J, Shetty Anala V, Liang Shun-Qing, Steer Clifford J
Department of Medicine, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA.
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA.
Genes (Basel). 2025 Jul 15;16(7):825. doi: 10.3390/genes16070825.
While prime editing offers improved precision compared to traditional CRISPR-Cas9 systems, concerns remain regarding potential off-target effects, including epigenetic changes such as DNA methylation. In this study, we investigated whether prime editing induces aberrant CpG methylation patterns. Whole-genome bisulfite sequencing revealed overall methylation similarity between Cas9-edited, and PE2-edited cells. However, localized epigenetic changes were observed, particularly in CpG islands and exon regions. The PE2-edited group showed a higher proportion of differentially methylated regions (DMRs) in some coding sequences compared to controls and Cas9-edited samples. Notably, CpG island methylation reached 0.18% in the PE2 vs. Cas9 comparison, indicating a higher susceptibility of these regulatory elements to epigenetic alterations by prime editing. Molecular function analyses including Gene Ontology and KEGG pathway analyses further revealed enrichment in molecular functions related to transcriptional regulation and redox activity in PE2-edited cells. These findings suggest that prime editing, while precise, may introduce subtle but functionally relevant methylation changes that could influence gene expression and cellular pathways. In summary, prime editing can induce localized DNA methylation changes in human cells, particularly within regulatory and coding regions. Understanding these epigenetic consequences is critical for the development of safer and more effective therapeutic applications of genome editing technologies.
与传统的CRISPR-Cas9系统相比，碱基编辑提高了精确性，但人们仍然担心其潜在的脱靶效应，包括DNA甲基化等表观遗传变化。在本研究中，我们调查了碱基编辑是否会诱导异常的CpG甲基化模式。全基因组亚硫酸氢盐测序揭示了Cas9编辑细胞和PE2编辑细胞之间的整体甲基化相似性。然而，观察到了局部表观遗传变化，特别是在CpG岛和外显子区域。与对照组和Cas9编辑样本相比，PE2编辑组在一些编码序列中显示出更高比例的差异甲基化区域（DMR）。值得注意的是，在PE2与Cas9的比较中，CpG岛甲基化达到0.18%，表明这些调控元件对碱基编辑引起的表观遗传改变更敏感。包括基因本体论和KEGG通路分析在内的分子功能分析进一步揭示了PE2编辑细胞中与转录调控和氧化还原活性相关的分子功能富集。这些发现表明，碱基编辑虽然精确，但可能会引入细微但功能相关的甲基化变化，从而影响基因表达和细胞通路。总之，碱基编辑可以在人类细胞中诱导局部DNA甲基化变化，特别是在调控和编码区域内。了解这些表观遗传后果对于开发更安全、更有效的基因组编辑技术治疗应用至关重要。
