In Vivo Knockout of the Vegfa Gene by Lentiviral Delivery of CRISPR/Cas9 in Mouse Retinal Pigment Epithelium Cells.

ABSTRACT: Virus-based gene therapy by CRISPR/Cas9-mediated genome editing and knockout may provide a new option for treatment of inherited and acquired ocular diseases of the retina. In support of this notion, we show that Streptococcus pyogenes (Sp) Cas9, delivered by lentiviral vectors (LVs), can be used in vivo to selectively ablate the vascular endothelial growth factor A (Vegfa) gene in mice. By generating LVs encoding SpCas9 targeted to Vegfa, and in parallel the fluorescent eGFP marker protein, we demonstrate robust knockout of Vegfa that leads to a significant reduction of VEGFA protein in transduced cells. Three of the designed single-guide RNAs (sgRNAs) induce in vitro indel formation at high frequencies (44%-93%). A single unilateral subretinal injection facilitates RPE-specific localizat

SUBMITTER: Holmgaard A 

PROVIDER: S-EPMC5626917 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature