The c.G6055A (p.G2019S) mutation in leucine-rich repeat kinase 2 (LRRK2) is the most prevalent genetic cause of Parkinson’s disease (PD). CRISPR/Cas9-mediated genome editing by homology-directed repair (HDR) has been applied to correct the mutation but may create small insertions and deletions (indels) due to double-strand DNA breaks. Adenine base editors (ABEs) could convert targeted A·T to G·C in genomic DNA without double-strand breaks. However, the correction efficiency of ABE in LRRK2 c.G6055A (p.G2019S) mutation remains unknown yet. This study aimed to compare the mutation correction efficiencies and off-target effects between HDR and ABEs in induced pluripotent stem cells (iPSCs) carrying LRRK2 c.G6055A (p.G2019S) mutation. A set of mutation-corrected isogenic lines by editing the LRRK2 c.G6055A (p.G2019S) mutation in a PD patient-derived iPSC line using HDR or ABE were established. The mutation correction efficacies, off-target effects, and indels between HDR and ABE were compared. Comparative transcriptomic and proteomic analyses between the LRRK2 p.G2019S iPSCs and isogenic control cells were performed to identify novel molecular targets involved in LRRK2-parkinsonism pathways. ABE had a higher correction rate (13/53 clones, 24.5%) than HDR (3/47 clones, 6.4%). Twenty-seven HDR clones (57.4%), but no ABE clones, had deletions, though 14 ABE clones (26.4%) had off-target mutations. The corrected isogenic iPSC-derived dopaminergic neurons exhibited reduced LRRK2 kinase activity, decreased phospho-α-synuclein expression, and mitigated neurite shrinkage and apoptosis. Comparative transcriptomic and proteomic analysis identified different gene expression patterns in energy metabolism, protein degradation, and peroxisome proliferator-activated receptor pathways between the mutant and isogenic control cells. The results of this study envision that ABE could directly correct the pathogenic mutation in iPSCs for reversing disease-related phenotypes in neuropathology and exploring novel pathophysiological targets in PD.

中文翻译：

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体外基因组编辑拯救携带 LRRK2 p.G2019S 突变的诱导多能干细胞衍生的多巴胺能神经元中的帕金森症表型

富含亮氨酸的重复激酶 2 (LRRK2) 中的 c.G6055A (p.G2019S) 突变是帕金森病 (PD) 最普遍的遗传原因。通过同源定向修复 (HDR) 进行的 CRISPR/Cas9 介导的基因组编辑已被用于纠正突变，但由于双链 DNA 断裂，可能会产生小的插入和缺失（插入缺失）。腺嘌呤碱基编辑器 (ABE) 可以将基因组 DNA 中的靶向 A·T 转化为 G·C，而不会出现双链断裂。然而，ABE 在 LRRK2 c.G6055A (p.G2019S) 突变中的校正效率仍然未知。本研究旨在比较 HDR 和 ABE 在携带 LRRK2 c.G6055A (p.G2019S) 突变的诱导多能干细胞 (iPSC) 中的突变校正效率和脱靶效应。通过编辑 LRRK2 c.G6055A (p. 使用 HDR 或 ABE 在 PD 患者衍生的 iPSC 系中建立了 G2019S) 突变。比较了 HDR 和 ABE 之间的突变校正效果、脱靶效应和插入缺失。在 LRRK2 p.G2019S iPSCs 和同基因对照细胞之间进行了比较转录组学和蛋白质组学分析，以确定参与 LRRK2-帕金森病通路的新分子靶点。ABE 的校正率（13/53 克隆，24.5%）高于 HDR（3/47 克隆，6.4%）。27 个 HDR 克隆（57.4%）有缺失，但没有 ABE 克隆，但 14 个 ABE 克隆（26.4%）有脱靶突变。校正后的同基因 iPSC 衍生的多巴胺能神经元表现出 LRRK2 激酶活性降低，磷酸-α-突触核蛋白表达降低，并减轻神经突收缩和细胞凋亡。比较转录组学和蛋白质组学分析确定了突变体和同基因对照细胞之间能量代谢、蛋白质降解和过氧化物酶体增殖物激活受体途径中的不同基因表达模式。本研究结果设想 ABE 可以直接纠正 iPSC 中的致病突变，从而逆转神经病理学中与疾病相关的表型，并探索 PD 中的新病理生理学靶点。