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Using CRISPR/Cas9 to achieve allele-specific editing of mutant Crx in a mouse model of Leber congenital amaurosis

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Posterboard#: A0277

Abstract Number: 4956 - A0277

AuthorBlock: Kathleen R. Chirco1, Shereen Chew1, Jacque L. Duncan1, Deepak A. Lamba1,2
1Department of Ophthalmology, University of California San Francisco, San Francisco, California, United States; 2Buck Institute for Research on Aging, Novato, California, United States;

DisclosureBlock: Kathleen R. Chirco, None; Shereen Chew, None; Jacque L. Duncan, None; Deepak A. Lamba, None;

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.

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