Reduced Expression of VEGF-A in Human Retinal Pigment Epithelial Cells and Human Muller Cells Following CRISPR-Cas9 Ribonucleoprotein-Mediated Gene Disruption.

ABSTRACT:

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% i

SUBMITTER: Ameri H 

PROVIDER: S-EPMC7422915 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature