June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Developing a Zebrafish Model of Dominant Optic Atrophy
Author Affiliations & Notes
  • Elin Strachan
    SBBS, University College Dublin, Dublin, Ireland
  • Breandan N Kennedy
    SBBS, University College Dublin, Dublin, Ireland
  • Niamh O'Sullivan
    SBBS, University College Dublin, Dublin, Ireland
  • Footnotes
    Commercial Relationships   Elin Strachan None; Breandan Kennedy None; Niamh O'Sullivan None
  • Footnotes
    Support  Irish Research Council GOIPG/2020/1312
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1589 – A0378. doi:
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      Elin Strachan, Breandan N Kennedy, Niamh O'Sullivan; Developing a Zebrafish Model of Dominant Optic Atrophy. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1589 – A0378.

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

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Abstract

Purpose : Optic Atrophy (OA) is a hereditary neurodegenerative disease characterised by progressive sight loss. This is associated with the irreversible degeneration of retinal ganglion cells. Most cases are associated with dominant mutations in the gene OPA1 (optic atrophy 1), a protein essential for mitochondrial fission – many OA patient fibroblasts have previously been noted to have varying mitochondrial dysfunction. It is unclear why mitochondrial dysfunction leads to retinal ganglion cell death, and there is currently no treatment for OA. I aim to create a zebrafish model of optic atrophy in order to understand the pathogenic mechanisms leading to sight loss in this condition.

Methods : I have designed pairs CRISPR guides to target commonly mutated regions of the zebrafish Opa1 gene, to create a large deletion. This deletion is predicted to cause a loss of function in the resulting protein. These guides were injected into 1-4 cell stage zebrafish embryos, and I subsequently performed optokinetic response assays to determine the presence of a visual defect in the resulting crispants.

Results : I was able to validate the presence of the predicted deletion through PCR and sequencing. Optokinetic response assays revealed a significant reduction in visual acuity in 5dpf crispants compared to siblings without deletion events. I am in the process of raising a subset of the crispants to produce a stable heterozygous line to repeat and validate these experiments.

Conclusions :
I have designed and validated CRISPR guides to create a large deletion in the Opa1 gene. Crispants injected with these guides display a reduction in visual acuity consistent with sight loss seen in patients with loss of OPA1 function. This is a positive early indication this model could be used to study the pathogenicity of loss of function Opa1 mutations. The visual phenotype could additionally be used to screen for modulators of Opa1 function in future.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

An overview of the workflow to produce crispants. 1-4 cell stage embryos are injected with a pair of crRNA guides complementary for regions in exons 8 and 9, which are commonly mutated in Opa1 patients. This creates 2 double strand breaks at each location, which creates a large deletion through non-homologous end joining.

An overview of the workflow to produce crispants. 1-4 cell stage embryos are injected with a pair of crRNA guides complementary for regions in exons 8 and 9, which are commonly mutated in Opa1 patients. This creates 2 double strand breaks at each location, which creates a large deletion through non-homologous end joining.

 

Zebrafish larvae with deletions in the Opa1 gene have a significant reduction in optokinetic response compared to their siblings. This is indicative of a reduction in visual acuity.

Zebrafish larvae with deletions in the Opa1 gene have a significant reduction in optokinetic response compared to their siblings. This is indicative of a reduction in visual acuity.

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