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利用先进的基因组编辑工具精确校正导致视网膜变性的-L659P突变并使旁观者编辑降至最低

Precise Correction of the -L659P Mutation Causing Retinal Degeneration with Minimum Bystander Editing by Advanced Genome Editing Tools.

作者信息

Liu Zhiquan, Chen Siyu, Sun Yang

机构信息

Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, CA 94304, USA.

Palo Alto Veterans Administration, Palo Alto, CA, USA.

出版信息

Research (Wash D C). 2025 Jul 2;8:0770. doi: 10.34133/research.0770. eCollection 2025.


DOI:10.34133/research.0770
PMID:40607323
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12220932/
Abstract

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.

摘要

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。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d54/12220932/adab79cb4a37/research.0770.fig.007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d54/12220932/69e702b505ed/research.0770.fig.001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d54/12220932/a464a13def20/research.0770.fig.002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d54/12220932/9669e07eb015/research.0770.fig.003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d54/12220932/28473491e7dd/research.0770.fig.004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d54/12220932/1eff757a3364/research.0770.fig.005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d54/12220932/bca9ba791b5d/research.0770.fig.006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d54/12220932/adab79cb4a37/research.0770.fig.007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d54/12220932/69e702b505ed/research.0770.fig.001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d54/12220932/a464a13def20/research.0770.fig.002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d54/12220932/9669e07eb015/research.0770.fig.003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d54/12220932/28473491e7dd/research.0770.fig.004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d54/12220932/1eff757a3364/research.0770.fig.005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d54/12220932/bca9ba791b5d/research.0770.fig.006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d54/12220932/adab79cb4a37/research.0770.fig.007.jpg

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本文引用的文献

[1]
Bystander editing by adenine base editors impairs vision restoration in a mouse model of Leber congenital amaurosis.

Mol Ther Methods Clin Dev. 2025-4-2

[2]
Engineered nucleocytosolic vehicles for loading of programmable editors.

Cell. 2025-5-15

[3]
In vivo prime editing rescues photoreceptor degeneration in nonsense mutant retinitis pigmentosa.

Nat Commun. 2025-3-10

[4]
Delivery of Prime editing in human stem cells using pseudoviral NanoScribes particles.

Nat Commun. 2025-1-4

[5]
Safer and efficient base editing and prime editing via ribonucleoproteins delivered through optimized lipid-nanoparticle formulations.

Nat Biomed Eng. 2025-1

[6]
Efficient Rescue of Retinal Degeneration in Pde6a Mice by Engineered Base Editing and Prime Editing.

Adv Sci (Weinh). 2024-11

[7]
Precise and versatile genome editing with click editors.

Nat Biotechnol. 2024-7-22

[8]
Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases.

Nat Biotechnol. 2024-7-22

[9]
Machine learning prediction of prime editing efficiency across diverse chromatin contexts.

Nat Biotechnol. 2025-5

[10]
Unlocking the Therapeutic Applicability of LNP-mRNA: Chemistry, Formulation, and Clinical Strategies.

Research (Wash D C). 2024-6-18

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