Prime Editing of Vascular Endothelial Growth Factor Receptor 2 Attenuates Angiogenesis In Vitro.

IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY CRISPR Journal Pub Date : 2024-08-01 Epub Date: 2024-08-07 DOI:10.1089/crispr.2024.0019
Gaoen Ma, Hui Qi, Hongwei Deng, Lijun Dong, Qing Zhang, Junkai Ma, Yanhui Yang, Xiaohe Yan, Yajian Duan, Hetian Lei
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Abstract

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) MutL homolog 1 gene with nicking guide RNA. PE6h was used to edit VEGFR2 (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 in vitro. Overall, our results highlight the potential of PE6h to inhibit angiogenesis in vivo.

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血管内皮生长因子受体 2 的基因编辑抑制体外血管生成
血管内皮生长因子受体(VEGFR)-2 是血管生成的关键开关,可在多种人类疾病中观察到。本研究开发了一种新型的高级质粒编辑(PE)系统,称为 PE6h,由双慢病毒载体组成:(1)融合了反转录酶和增强型 PE 引导 RNA 的簇状规则间隔 palindromic repeat-associated protein 9 (H840A)切口酶;(2)带有切口引导 RNA 的显性阴性 (DN) MutL 同源物 1 基因。利用 PE6h 编辑 VEGFR2(c.18315T>A,50.8%),生成一个过早终止密码子(从 AAG 变为 TAG),从而在人视网膜微血管内皮细胞(HRECs)中产生 DN-VEGFR2(787 aa)。DN-VEGFR2 阻碍了 VEGF 诱导的 VEGFR2、Akt 和细胞外信号调节激酶-1/2 的磷酸化,并阻碍了 PE6h 修饰的 HRECs 体外管形成。总之,我们的研究结果凸显了 PE6h 抑制体内血管生成的潜力。
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来源期刊
CRISPR Journal
CRISPR Journal Biochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
6.30
自引率
2.70%
发文量
76
期刊介绍: In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR. Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.
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