July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Using CRISPR/Cas9 to achieve allele-specific editing of mutant Crx in a mouse model of Leber congenital amaurosis
Author Affiliations & Notes
  • Kathleen R Chirco
    Department of Ophthalmology, University of California San Francisco, San Francisco, California, United States
  • Shereen Chew
    Department of Ophthalmology, University of California San Francisco, San Francisco, California, United States
  • Jacque L Duncan
    Department of Ophthalmology, University of California San Francisco, San Francisco, California, United States
  • Deepak A Lamba
    Department of Ophthalmology, University of California San Francisco, San Francisco, California, United States
    Buck Institute for Research on Aging, Novato, California, United States
  • Footnotes
    Commercial Relationships   Kathleen Chirco, None; Shereen Chew, None; Jacque Duncan, None; Deepak Lamba, None
  • Footnotes
    Support  NIH grant EY025779
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4956. doi:
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    • Get Citation

      Kathleen R Chirco, Shereen Chew, Jacque L Duncan, Deepak A Lamba; Using CRISPR/Cas9 to achieve allele-specific editing of mutant Crx in a mouse model of Leber congenital amaurosis. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4956.

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

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Abstract

Purpose : Dominant cone-rod homeobox (CRX)-associated Leber congenital amaurosis (LCA) is a severe retinal degenerative disease for which no treatments are currently available. The goal of this study was to utilize allele-specific CRISPR/Cas9 editing to alleviate LCA-associated photoreceptor phenotypes in mice with dominant variants in Crx.

Methods : Mouse embryonic fibroblasts (MEFs) were collected from an LCA mouse model, CrxE168d2/d2, along with CrxE168d2/+, and C57BL6/J mice. MEFs from all three lines were reprogrammed using the Sendai virus (MOI 5-5-3) to generate mouse induced pluripotent stem cell (miPSC) lines. After 25 days, stem cell-like colonies were picked and passaged up to 10 times to ensure complete reprogramming. Immunocytochemistry and qPCR were performed to confirm pluripotency for each miPSC line (n=3 per mouse), using antibodies or primers targeting Nanog, Oct4, and Sox2. Up to 20 guide RNA (gRNA) molecules targeting the mutant Crx allele were designed for each mouse line. Each gRNA was tested for cutting efficiency using a T7 endonuclease assay.

Results : MEF lines from CrxE168d2/d2, CrxE168d2/+, and C57BL6/J mice were successfully reprogrammed to generate stable miPSC colonies (n=2 per mouse). Immunocytochemistry and qPCR analysis to verify reprogramming of each miPSC line revealed the presence of key pluripotency markers, Nanog, Oct4, and Sox2, in each line (n=3 per mouse). A T7 endonuclease assay demonstrated CRISPR-mediated editing events within the Crx gene for various gRNAs tested, and three of the most successful gRNAs were validated by sequencing.

Conclusions : IPSCs provide us with a valuable in vitro model system to study the development of LCA. Future work will include the generation of retinal organoids from each miPSC line, which will allow us to compare photoreceptor development from CRISPR/Cas9-edited versus un-edited miPSCs.

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

 

Mouse embryonic fibroblast (MEF) isolation and reprogramming
MEFs were immunolabeled with anti-vimentin (green) and anti-TUJ1 (red) antibodies (A), reprogrammed colonies were picked and expanded (B), MEF DNA was sequenced to identify allelic variants (C), and miPSC colonies were immunolabeled with anti-Sox2 (green) and anti-Oct4 (red) antibodies (D-G). Scale bar = 50µm.

Mouse embryonic fibroblast (MEF) isolation and reprogramming
MEFs were immunolabeled with anti-vimentin (green) and anti-TUJ1 (red) antibodies (A), reprogrammed colonies were picked and expanded (B), MEF DNA was sequenced to identify allelic variants (C), and miPSC colonies were immunolabeled with anti-Sox2 (green) and anti-Oct4 (red) antibodies (D-G). Scale bar = 50µm.

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