July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Novel Zebrafish Autosomal Recessive Retinitis Pigmentosa Disease Models Created by CRISPR/Cas9 Gene Editing
Author Affiliations & Notes
  • Liyun Zhang
    Wilmer Eye Inst-Smith Bldg Rm 4001, Johns Hopkins School of Medicine, Baltimore, Maryland, United States
  • Arife Unal Eroglu
    Wilmer Eye Inst-Smith Bldg Rm 4001, Johns Hopkins School of Medicine, Baltimore, Maryland, United States
  • Timothy Mulligan
    Wilmer Eye Inst-Smith Bldg Rm 4001, Johns Hopkins School of Medicine, Baltimore, Maryland, United States
  • Jie Dong
    Wilmer Eye Inst-Smith Bldg Rm 4001, Johns Hopkins School of Medicine, Baltimore, Maryland, United States
  • Emilie Cheng
    Wilmer Eye Inst-Smith Bldg Rm 4001, Johns Hopkins School of Medicine, Baltimore, Maryland, United States
  • Naveena Murugan
    Wilmer Eye Inst-Smith Bldg Rm 4001, Johns Hopkins School of Medicine, Baltimore, Maryland, United States
  • Wuhong Pei
    National Human Genome Research Institute, Bethesda, Maryland, United States
  • Lisha Xu
    National Human Genome Research Institute, Bethesda, Maryland, United States
  • Shawn Burgess
    National Human Genome Research Institute, Bethesda, Maryland, United States
  • Meera Saxena
    Wilmer Eye Inst-Smith Bldg Rm 4001, Johns Hopkins School of Medicine, Baltimore, Maryland, United States
  • Jeff S Mumm
    Wilmer Eye Inst-Smith Bldg Rm 4001, Johns Hopkins School of Medicine, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Liyun Zhang, None; Arife Unal Eroglu, None; Timothy Mulligan, None; Jie Dong, None; Emilie Cheng, None; Naveena Murugan, None; Wuhong Pei, None; Lisha Xu, None; Shawn Burgess, None; Meera Saxena, None; Jeff Mumm, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 460. doi:
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      Liyun Zhang, Arife Unal Eroglu, Timothy Mulligan, Jie Dong, Emilie Cheng, Naveena Murugan, Wuhong Pei, Lisha Xu, Shawn Burgess, Meera Saxena, Jeff S Mumm; Novel Zebrafish Autosomal Recessive Retinitis Pigmentosa Disease Models Created by CRISPR/Cas9 Gene Editing. Invest. Ophthalmol. Vis. Sci. 2019;60(9):460.

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

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Abstract

Purpose : Retinitis pigmentosa (RP) is a group of inherited retinal degeneration diseases which cause irreversible vision loss. Of ~90 genes associated with RP to date, underlying disease mechanisms are often unclear. To better understand RP-linked gene function and create novel disease models, we utilized CRISPR/Cas9-based genome editing technology to disrupt homologs of RP-linked genes in zebrafish.

Methods : Eight zebrafish homologs of five human genes linked to autosomal recessive RP (PDE6A, PDE6B, PDE6G, ABCA4 and RHO) were selected for CRISPR/Cas9 targeting. One or two guide RNAs targeting the 5’-end of each gene were co-injected with Cas9 mRNA into zebrafish embryos at the one cell stage. To facilitate phenotyping, genotyped founders (F0) were crossed to a transgenic line in which rod photoreceptors are labeled with YFP. F1 progeny carrying predicted loss-of-function frameshift mutations were pair mated, YFP+ offspring collected, and YFP expression levels monitored from 4 to 8 days post-fertilization using fluorescence microscopy. At day 8, F2 progeny were split into high and low expressing YFP+ groups. Representative larvae from each group were genotyped and confocal imaging performed to assess changes in rod cell numbers and morphology.

Results : All eight genes were successfully targeted. Predicted frameshift mutations were identified in all genes. Here, we report on novel phosphodiesterase 6b (pde6b) autosomal recessive mutants and autosomal dominant and recessive rhodopsin (rho) mutants. Decreased YFP expression was observed to be highly associated with mutant genotypes. Confocal imaging of mutant fish from 4 to 8 dpf confirmed the loss of rod photoreceptors, a hallmark of RP. Interestingly, autosomal dominant rho mutants undergo severe rod cell loss at early developmental stages, while recessive rho and pde6b mutants exhibit gradual rod cell loss.

Conclusions : In summary, we demonstrate a simple transgene-assisted screening method for identifying RP-like phenotypes in larval zebrafish. Novel zebrafish lines carrying mutations in RP-implicated genes can serve as models for future studies focusing on disease gene mechanisms and for large-scale drug screening.

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

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