September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Genomic Disruption of VEGF in Human Retinal Pigment Epithelial Cells using CRISPR-Cas9 Endonuclease.
Author Affiliations & Notes
  • Glenn Yiu
    Dept of Ophthalmology, University of California, Davis, Sacramento, California, United States
  • Eric Tieu
    Dept of Ophthalmology, University of California, Davis, Sacramento, California, United States
  • Brittany Wong
    Duke University, Durham, North Carolina, United States
  • Anthony Nguyen
    Dept of Ophthalmology, University of California, Davis, Sacramento, California, United States
  • Zeljka Smit-McBride
    Dept of Ophthalmology, University of California, Davis, Sacramento, California, United States
  • Footnotes
    Commercial Relationships   Glenn Yiu, None; Eric Tieu, None; Brittany Wong, None; Anthony Nguyen, None; Zeljka Smit-McBride, None
  • Footnotes
    Support  UC Davis Center for Vision Science Seed Grant
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 1159. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Glenn Yiu, Eric Tieu, Brittany Wong, Anthony Nguyen, Zeljka Smit-McBride; Genomic Disruption of VEGF in Human Retinal Pigment Epithelial Cells using CRISPR-Cas9 Endonuclease.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1159.

      Download citation file:


      © 2017 Association for Research in Vision and Ophthalmology.

      ×
  • Supplements
Abstract

Purpose : To evaluate the use of type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease as a potential form of gene therapy for ocular angiogenesis by genomic disruption of vascular endothelial growth factor (VEGF-A) in human retinal pigment epithelial (RPE) cells.

Methods : CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified using 3 different algorithms for predicting on-target and off-target probabilities. Single-guide RNA (sgRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the S. pyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect monolayers of cultured ARPE-19 cells, a human RPE cell line, for 7 days with puromycin to select for transduced cells. Frequency of insertion or deletion (indel) mutations was assessed by T7E1 mismatch detection assay, mRNA levels were measured by quantitative RT-PCR, and protein levels were determined by enzyme-linked immunosorbent assay (ELISA) for secreted VEGF-A. Cells infected with lentivirus expressing SpCas9 only without sgRNA were used as controls. The T7E1 assay was used to determine indel frequency at the top 3 putative off-target sites for the CRISPR sequences with the highest Cas9 activities.

Results : A total of 6 sgRNAs 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 all 6 sgRNAs with SpCas9 resulted in indel formation in the VEGF-A gene, with frequencies ranging from 33.8 ± 5.4 % to 35.6 ± 2.6 %. One sgRNA resulted in a 1.56 ± 0.31 fold reduction in VEGF-A mRNA levels as compared with SpCas9-only control (P=0.005), using GADPH, HPRT1, and beta-2 microtubulin as reference genes. There was a corresponding decrease in secreted VEGF-A protein of 19.9 ± 2.0 % (P = 0.04). No significant indel formation in the top 3 putative off-target sites were detected, suggesting that the CRISPR-Cas9-mediated knockdown of VEGF-A is specific.

Conclusions : The CRISPR-Cas9 endonuclease system may reduce VEGF-A expression and secretion from human RPE cells, supporting the possibility of employing genome engineering for treatment of neovascular retinal diseases.

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

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×