July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Development of a CRISPR dCas9-KRAB based strategy for treatment for dominant retinal degenerative blindness
Author Affiliations & Notes
  • Erin R. Burnight
    Institute for Vision Research, Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Jessica A. Cooke
    Institute for Vision Research, Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Darcey Klaahsen
    Institute for Vision Research, Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Luan M. Streb
    Institute for Vision Research, Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Louisa M. Affatigato
    Institute for Vision Research, Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Robert F Mullins
    Institute for Vision Research, Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Edwin M Stone
    Institute for Vision Research, Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Budd Tucker
    Institute for Vision Research, Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States
  • Footnotes
    Commercial Relationships   Erin Burnight, None; Jessica Cooke, None; Darcey Klaahsen, None; Luan Streb, None; Louisa Affatigato, None; Robert Mullins, None; Edwin Stone, None; Budd Tucker, None
  • Footnotes
    Support   R01EY024588; R01EY026008
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 5983. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Erin R. Burnight, Jessica A. Cooke, Darcey Klaahsen, Luan M. Streb, Louisa M. Affatigato, Robert F Mullins, Edwin M Stone, Budd Tucker; Development of a CRISPR dCas9-KRAB based strategy for treatment for dominant retinal degenerative blindness. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5983.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose : Inherited retinal disease is a major cause of blindness worldwide. In a recent survey of one thousand consecutive families clinically diagnosed with an inherited eye disease, the disease causing genotype was identified in 76% of cases. Of these, approximately 12% carried dominantly inherited variants, of which approximately 20% lay within the gene BEST1. Expression of BEST1is restricted to RPE cells, and dominant mutations in BEST1 cause a maculopathy characterized by excessive accumulation of autofluorescent material between the RPE cells and photoreceptor outer segments leading to formation of subretinal lesions and loss of central vision. Treatment for this and other dominantly inherited retinal dystrophies may benefit from knockdown or ablation of mutant allele expression. To that end the purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy capable of suppressingBEST1expression.

Methods : We created CRISPR-Cas9 transcriptional knockdown reagents using catalytically inactive S. pyogenes Cas9 (dCas9) fused to the Krüppel associated box (KRAB) transcriptional repressor domain and evaluated six guides targeted to transcriptional regulatory elements in the promoter region of BEST1. Using a reporter construct carrying GFP cloned downstream of the BEST1 promoter fragment (nucleotides -600 to +38), we assayed GFP knockdown in cells treated with our BEST1promoter-targeted CRISPR-dCas9 reagents. Following functional confirmation, reagents were packaged into lentiviral particles for targeting of patient derived RPE cells (iPSC-RPE).

Results : Quantitative RT-PCR analysis of cells treated with CRISPR-dCas9 and reporter plasmids demonstrated ~70%-79% reduction in GFP expression when compared to control cells treated with reporter plasmid only. Two guides with the greatest knockdown in this assay (g465 and g539) were cloned into lentiviral transgene cassette plasmids along with dCas9-KRAB, packaged into VSVG-pseudotyped lentiviral particles. To assess gene knockdown in a disease specific model system, iPSC derived RPE cells were generated.

Conclusions : We have generated a dCas9-KRAB gene repression system suitable for suppressing BEST1expression. This work may provide a paradigm from which to develop CRISPR-dCas9 therapies to treat dominantly inherited retinal dystrophies.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

×
×

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.

×