Department of Ophthalmology, The First Affiliated Hospital of Hainan Medical University, Haikou, China.
Department of Ophthalmology, The Third Affiliated Hospital of Xinxiang Medical University, Xinxiang, China.
CRISPR J. 2024 Aug;7(4):188-196. doi: 10.1089/crispr.2024.0019. Epub 2024 Aug 7.
Vascular endothelial growth factor receptor (VEGFR)-2 is a key switch for angiogenesis, which is observed in various human diseases. In this study, a novel system for advanced prime editing (PE), termed PE6h, is developed, consisting of dual lentiviral vectors: (1) a clustered regularly interspaced palindromic repeat-associated protein 9 (H840A) nickase fused with reverse transcriptase and an enhanced PE guide RNA and (2) a dominant negative (DN) homolog 1 gene with nicking guide RNA. PE6h was used to edit (c.18315T>A, 50.8%) to generate a premature stop codon (TAG from AAG), resulting in the production of DN-VEGFR2 (787 aa) in human retinal microvascular endothelial cells (HRECs). DN-VEGFR2 impeded VEGF-induced phosphorylation of VEGFR2, Akt, and extracellular signal-regulated kinase-1/2 and tube formation in PE6h-edited HRECs . Overall, our results highlight the potential of PE6h to inhibit angiogenesis .
血管内皮生长因子受体 (VEGFR)-2 是血管生成的关键开关，在各种人类疾病中都有观察到。在这项研究中，开发了一种新型的高级 Prime 编辑 (PE) 系统，称为 PE6h，它由双慢病毒载体组成：(1) 带有逆转录酶和增强型 PE 指导 RNA 的成簇规律间隔短回文重复相关蛋白 9 (H840A) 切口酶与 (2) 带有切口指导 RNA 的显性负 (DN) 同源 1 基因。PE6h 用于编辑 (c.18315T>A, 50.8%)，以产生一个提前终止密码子 (TAG 取代 AAG)，导致 DN-VEGFR2（787 aa）在人视网膜微血管内皮细胞 (HRECs) 中产生。DN-VEGFR2 阻止了 VEGF 诱导的 VEGFR2、Akt 和细胞外信号调节激酶-1/2 的磷酸化以及 PE6h 编辑的 HRECs 中的管形成。总体而言，我们的结果强调了 PE6h 抑制血管生成的潜力。
