Purchase this article with an account.
Xionggao Huang, Gaoen Ma, Yewlin Chee, Shizuo Mukai, Patricia A. D’Amore, Hetian Lei; Inactivation of VEGFR2 using CRISPR/Cas9 provides superior inhibition to the anti-VEGF drugs. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5024.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Inhibitors such as Lucentis and Eylea have become important therapies for ocular angiogenesis. While these drugs are effective, continuous ocular injections are required. CRISPR-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) system has been demonstrated to be a simple and efficient tool for genome editing and protein depletion in cultured human cells and mice. The goal of this project was to explore a novel approach for blocking the signaling transduction from VEGF to VEGFR2 using CRISPR/Cas9 technology.
Four guide RNAs (gRNA) (k11-k14) based on exon 2 of human VEGFR2 genomic sequence (NM_002253.2) were selected (http://crispr.mit.edu/), Control sgRNA sequence was designed to target lacZ gene from E. coli. These were cloned into the lentiv2-Cas9 vector (Addgene:52961), respectively, and were confirmed by DNA sequencing. Lentiviruses produced in293T cells were used to infect porcine aortic endothelial cells overexpressing VEGFR2 (PAEC-KDR). The DNA fragments around the expected Cas9 cleavage site were analyzed by Sanger sequencing, surveyor nuclease assay and next generation sequencing (NGS). The efficiency of CRISPR/Cas9-mediated VEGFR2 depletion and VEGF-stimulated phosphorylation of VEGFR2, Akt and Erk were determined by western blot.
Sanger DNA sequencingand surveyor nuclease assay demonstrated that VEGFR2 gene was successfully edited by SpCas9 guided by the gRNA k12, and NGS indicated there were two major mutated sequences (42.37% and 22.53%) among the 11 various sequences. Depletion of VEGFR2 in the cells with SpCas9-VEGFR2 gRNA k12 was confirmed by a western blotting analysis. The VEGFR2-depleted cells had lower levels of Akt and Erk phosphorylation than the LacZ control cells treated with Lucentis or Eylea.
The depletion of VEGFR2 using the CRISPR/Cas9 technology and CRISPR/Cas9 mediated VEGFR2 genomic gene silencing was superior to the anti-VEGF drugs (Lucentis and Eylea) for suppressing VEGF-stimulated signaling pathway. The CRISPR-Cas9 technology provides a novel opportunity to inhibit pathological angiogenesis.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
This PDF is available to Subscribers Only