Reduced Expression of VEGF-A in Human Retinal Pigment Epithelial Cells and Human Muller Cells Following CRISPR-Cas9 Ribonucleoprotein-Mediated Gene Disruption
Reduced Expression of VEGF-A in Human Retinal Pigment Epithelial Cells and Human Muller Cells Following CRISPR-Cas9 Ribonucleoprotein-Mediated Gene Disruption
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DOI：
10.1167/tvst.9.8.23
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发表时间：
2020-07-01
影响因子：
3
通讯作者：
Hamm-Alvarez, Sarah F.
中科院分区：
文献类型：
--
作者：
Ameri, Hossein;Murat, Christopher;Hamm-Alvarez, Sarah F.
目的：评价血管内皮生长因子- a （VEGF-A）基因编辑对人眼主要VEGF-A产生细胞——视网膜色素上皮细胞（RPE）和Muller细胞的影响。方法：采用CRISPR-Cas9核糖核蛋白靶向VEGF-A基因外显子1。脂质体CRISPRMAX作为载体。对寡核苷酸和基因组dna进行体外基因编辑效率评估。采用Sanger测序检测索引。采用定量聚合酶链反应和酶联免疫吸附法检测VEGF-A信使RNA和蛋白的表达。结果：60个核苷酸DNA双链的体外切割实验显示，前体切割率为88%。RPE细胞的裂解效率为40%，Muller细胞的裂解效率为32%。在CRISPR-Cas9处理的RPE细胞和Muller细胞中，Sanger测序在两种细胞的预测切割位点上都显示了指数。VEGF-A基因破坏后，RPE细胞中VEGF-A蛋白水平下降43% (P < 0.0001)， Muller细胞中VEGF-A蛋白水平下降38% （P < 0.0001）。结论：crispr - cas9介导的基因破坏导致人RPE和Muller细胞中VEGF-A基因蛋白表达显著降低。CRISPR-Cas9核糖核蛋白可能允许同时靶向多个VEGF-A产生细胞。翻译相关性：使用CRISPR-Cas9核糖核蛋白破坏VEGF-A基因在治疗视网膜血管疾病方面具有潜力。
Purpose: To evaluate the effects of vascular endothelial growth factor-A (VEGF-A) gene editing in human retinal pigment epithelial (RPE) cells and human Muller cells, which are the main VEGF-A producing cells in the eye.Methods: CRISPR-Cas9 ribonucleoprotein was used to target exon 1 in VEGF-A gene. Lipofectamine CRISPRMAX was used as a vehicle. In vitro gene editing efficiency was assessed on oligonucleotides and genomic DNAs. Sanger sequencing was performed to detect indels. VEGF-A messenger RNA and protein expressions were assessed using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay.Results: In vitro cleavage assay on a 60-nucleotide DNA duplex showed 88% cleavage of the precursor. The cleavage efficiency was 40% in RPE cells and 32% in Muller cells. Sanger sequencing in the CRISPR-Cas9 treated RPE and Muller cells showed indels at the predicted cut site in both cells. After the VEGF-A gene disruption, VEGF-A protein levels decreased 43% in RPE cells (P < 0.0001) and 38% in Muller cells (P < 0.0001).Conclusions: CRISPR-Cas9-mediated gene disruption resulted in a significant decrease in the VEGF-A gene protein expression in human RPE and Muller cells. CRISPR-Cas9 ribonucleoprotein may allow simultaneous targeting of multiple VEGF-A producing cells.Translational Relevance: VEGF-A gene disruption using CRISPR-Cas9 ribonucleoprotein has a potential in treating retinal vascular diseases.