title: Editing VEGFR2 Blocks VEGF-Induced Activation of Akt and Tube Formation.
abstract: Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in VEGF-induced angiogenesis. The goal of this project was to test the hypothesis that editing genomic VEGFR2 loci using the technology of clustered regularly interspaced palindromic repeats (CRISPR)-associated DNA endonuclease (Cas)9 in Streptococcus pyogenes (SpCas9) was able to block VEGF-induced activation of Akt and tube formation.
  
  
  
  Four 20 nucleotides for synthesizing single-guide RNAs based on human genomic VEGFR2 exon 3 loci were selected and cloned into a lentiCRISPR v2 vector, respectively. The DNA fragments from the genomic VEGFR2 exon 3 of transduced primary human retinal microvascular endothelial cells (HRECs) were analyzed by Sanger DNA sequencing, surveyor nuclease assay, and next-generation sequencing (NGS). In the transduced cells, expression of VEGFR2 and VEGF-stimulated signaling events (e.g., Akt phosphorylation) were determined by Western blot analyses; VEGF-induced cellular responses (proliferation, migration, and tube formation) were examined.
  
  
  
  In the VEGFR2-sgRNA/SpCas9-transduced HRECs, Sanger DNA sequencing indicated that there were mutations, and NGS demonstrated that there were 83.57% insertion and deletions in the genomic VEGFR2 locus; expression of VEGFR2 was depleted in the VEGFR2-sgRNA/SpCas9-transduced HRECs. In addition, there were lower levels of Akt phosphorylation in HRECs with VEGFR2-sgRNA/SpCas9 than those with LacZ-sgRNA/SpCas9, and there was less VEGF-stimulated Akt activation, proliferation, migration, or tube formation in the VEGFR2-depleted HRECs than those treated with aflibercept or ranibizumab.
  
  
  
  The CRISPR-SpCas9 technology is a potential novel approach to prevention of pathologic angiogenesis.
authors.affiliation: Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States 2Massachusetts Eye & Ear, Harvard Medical School, Boston, Massachusetts, United States 3Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States 4Hainan Eye Hospital, Hainan Province, China.
authors.forename: Xionggao
authors.initials: X
authors.lastname: Huang
authors.affiliation: Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States 2Massachusetts Eye & Ear, Harvard Medical School, Boston, Massachusetts, United States 3Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: Guohong
authors.initials: G
authors.lastname: Zhou
authors.affiliation: Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States 2Massachusetts Eye & Ear, Harvard Medical School, Boston, Massachusetts, United States 3Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: Wenyi
authors.initials: W
authors.lastname: Wu
authors.affiliation: Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States 2Massachusetts Eye & Ear, Harvard Medical School, Boston, Massachusetts, United States 3Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: Gaoen
authors.initials: G
authors.lastname: Ma
authors.affiliation: Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States 2Massachusetts Eye & Ear, Harvard Medical School, Boston, Massachusetts, United States 3Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: D'Amore
authors.affiliation: Massachusetts Eye & Ear, Harvard Medical School, Boston, Massachusetts, United States 3Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: Shizuo
authors.initials: S
authors.lastname: Mukai
authors.affiliation: Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States 2Massachusetts Eye & Ear, Harvard Medical School, Boston, Massachusetts, United States 3Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
authors.forename: Hetian
authors.initials: H
authors.lastname: Lei
mesh_terms: D015153:Blotting, Western; D002465:Cell Movement / Q000502:physiology; D049109:Cell Proliferation / Q000502:physiology*; D002478:Cells, Cultured; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D003850:Deoxyribonuclease I / Q000235:genetics; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D000072669:Gene Editing / Q000379:methods*; D006801:Humans; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D013297:Streptococcus pyogenes / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D051057:Proto-Oncogene Proteins c-akt; D003850:Deoxyribonuclease I