September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Generation of a zebrafish model of achromatopsia using CRISPR/Cas9 genome editing
Author Affiliations & Notes
  • Michael D Varnum
    Integrative Physiology and Neuroscience, Washington State University, Pullman, Washington, United States
  • Tshering Sherpa
    Integrative Physiology and Neuroscience, Washington State University, Pullman, Washington, United States
  • Pete Meighan
    Integrative Physiology and Neuroscience, Washington State University, Pullman, Washington, United States
  • Footnotes
    Commercial Relationships   Michael Varnum, None; Tshering Sherpa, None; Pete Meighan, None
  • Footnotes
    Support  WSU/CVM Intramural and WSU-ADARP grants
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 81. doi:
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      Michael D Varnum, Tshering Sherpa, Pete Meighan; Generation of a zebrafish model of achromatopsia using CRISPR/Cas9 genome editing. Invest. Ophthalmol. Vis. Sci. 2016;57(12):81.

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

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Abstract

Purpose : There is a need for cone photoreceptor-rich animal models for cone-specific diseases, because of the relevance to human retinal degeneration involving loss of central, high-acuity vision. Inherited defects in the genes encoding human CNGA3 and CNGB3 have been linked to complete and incomplete achromatopsia, progressive cone dystrophy and macular degeneration. Here we have generated a new putative model for achromatopsia/cone dystrophy using CRISPR/Cas9 genome editing to target zebrafish CNGA3a.

Methods : We edited zebrafish CNGA3a using single-cell embryo microinjection of nls-zCas9-nls mRNA plus guide RNA(s) targeting sites within exon 9. For generation of a specific missense mutation, we also included a mutant oligonucleotide template for homology-directed repair, and SCR7 to chemically inhibit nonhomologous end-joining. Screening for CNGA3a editing was carried out using the PCR with oligos specific for mutant or wild-type sequences; mismatch cleavage using T7 endonuclease I; and/or DNA sequencing. The functional effects of CNGA3a editing were determined using optomotor response (OMR) visual performance tests and electroretinogram (ERG) recordings with 6-10 dpf larvae.

Results : Editing efficiency was approximately 7% for R356W mutation production and >50% for CNGA3a disruption by insertion or deletion (indel). CNGA3a indels were associated with disruption of visual function in larvae, as assessed via OMR tests and ERG recordings. Following Cas9/gRNA (CNGA3a) injections, surviving F0 larvae demonstrated impaired OMR performance: 46.2 +/- 7.7% OMR(+) compared to 89.6 +/- 3.6% OMR(+) and 95.0 +/- 5.8% OMR(+) for non-injected and mock-injected controls, respectively (p<0.01; N = 13- 16). In addition, OMR(-) larvae often presented attenuated ERG (b-wave) responses. The majority of OMR(-) larvae arising from injections for CNGA3a disruption were demonstrated to have predominant indels adjacent to one or both targeted PAM sites.

Conclusions : CRISPR/Cas9 editing of CNGA3a produced a decrement in visual function in F0 larvae. Genome editing of zebrafish CNGA3a (with or without CNGA3b editing) may ultimately present a model for human cone dysfunction/degeneration. Furthermore, functional screening at the larval stage may be of general use to enrich for CRISPR/Cas9-mediated disruption of cone phototransduction genes in zebrafish.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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