Liu Zhiquan, Chen Siyu, Sun Yang
Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, CA 94304, USA.
Palo Alto Veterans Administration, Palo Alto, CA, USA.
Research (Wash D C). 2025 Jul 2;8:0770. doi: 10.34133/research.0770. eCollection 2025.
Recently developed base editing (BE), prime editing (PE), and click editing (CE) technologies enable precise and efficient genome editing with minimal risk of double-strand breaks and associated toxicity. However, their effectiveness in correcting real disease-causing mutations has not been systematically compared. Here, we aim to evaluate the potential of BE, PE, and CE technologies in rescuing the retinal degeneration-causing (c.1976T>C, p.L659P) mutation. This site is prone to bystander effects, making it an ideal model for comparing the editing outcomes of these 3 novel technologies, particularly their editing precision. We optimized BE, PE, and CE systems in vitro using -L659P cell models and compared their editing via deep sequencing. BE and PE had similar efficiency, but PE was the most precise, minimizing bystander edits. CE had lower efficiency and higher indel rates, needing further optimization. Using the optimal PE system for in vivo electroporation in -L659P mice, we achieved 12.4% targeted repair with high precision, partially rescuing retinal degeneration. This study demonstrates proof of concept for the precise correction of the -L659P mutation causing retinal degeneration using BE, PE, and CE tools. The findings offer valuable insights into the future optimization of precision gene editing techniques and their potential translational applications.
最近开发的碱基编辑(BE)、引导编辑(PE)和点击编辑(CE)技术能够实现精确高效的基因组编辑,双链断裂及相关毒性风险最小。然而,它们在纠正实际致病突变方面的有效性尚未得到系统比较。在此,我们旨在评估BE、PE和CE技术在挽救导致视网膜变性的(c.1976T>C,p.L659P)突变方面的潜力。该位点容易产生旁观者效应,使其成为比较这三种新技术编辑结果,特别是其编辑精度的理想模型。我们使用-L659P细胞模型在体外优化了BE、PE和CE系统,并通过深度测序比较了它们的编辑情况。BE和PE效率相似,但PE最精确,将旁观者编辑降至最低。CE效率较低且插入缺失率较高,需要进一步优化。使用最佳的PE系统对-L659P小鼠进行体内电穿孔,我们实现了12.4%的高精度靶向修复,部分挽救了视网膜变性。本研究证明了使用BE、PE和CE工具精确纠正导致视网膜变性的-L659P突变的概念验证。这些发现为精确基因编辑技术的未来优化及其潜在的转化应用提供了有价值的见解。
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