title: Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease.
abstract: OBJECTIVE
  To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells.
  
  
  METHODS
  CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay.
  
  
  RESULTS
  Five gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% ± 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% ± 7.4% (P < 0.001), and reduction of endothelial tube formation up to 39.4% ± 9.8% (P = 0.02). No significant indel formation in the top three putative off-target sites tested was detected.
  
  
  CONCLUSIONS
  The CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases.
authors: Department of Ophthalmology & Vision Science, University of California, Davis, Sacramento, California, United States.
authors: Glenn
authors: G
authors: Yiu
authors: Department of Ophthalmology & Vision Science, University of California, Davis, Sacramento, California, United States.
authors: Eric
authors: E
authors: Tieu
authors: Department of Ophthalmology & Vision Science, University of California, Davis, Sacramento, California, United States.
authors: Anthony T
authors: AT
authors: Nguyen
authors: Duke University, Durham, North Carolina, United States.
authors: Brittany
authors: B
authors: Wong
authors: Department of Ophthalmology & Vision Science, University of California, Davis, Sacramento, California, United States.
authors: Zeljka
authors: Z
authors: Smit-McBride
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D000076987:CRISPR-Associated Protein 9; D002478:Cells, Cultured; D020256:Choroidal Neovascularization / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006801:Humans; D009154:Mutation; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D042461:Vascular Endothelial Growth Factor A; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases