June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Correction of an S Opsin Base-pair Deletion in Hamsters Using CRISPR-Cas9
Author Affiliations & Notes
  • Michelle M Giarmarco
    Ophthalmology, University of Washington, Seattle, Washington, United States
  • Alexandra F Neitz
    Molecular and Cellular Biology, University of Washington, Seattle, Washington, United States
  • James A Kuchenbecker
    Ophthalmology, University of Washington, Seattle, Washington, United States
  • Zhongde Wang
    Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah, United States
  • Yanan Liu
    Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah, United States
  • Rong Li
    Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah, United States
  • Jay Neitz
    Ophthalmology, University of Washington, Seattle, Washington, United States
  • Maureen Neitz
    Ophthalmology, University of Washington, Seattle, Washington, United States
  • Footnotes
    Commercial Relationships   Michelle Giarmarco None; Alexandra Neitz None; James Kuchenbecker None; Zhongde Wang None; Yanan Liu None; Rong Li None; Jay Neitz None; Maureen Neitz None
  • Footnotes
    Support  NIH NEI Grant P30-EY001730, Research to Prevent Blindness
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 761. doi:
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      Michelle M Giarmarco, Alexandra F Neitz, James A Kuchenbecker, Zhongde Wang, Yanan Liu, Rong Li, Jay Neitz, Maureen Neitz; Correction of an S Opsin Base-pair Deletion in Hamsters Using CRISPR-Cas9. Invest. Ophthalmol. Vis. Sci. 2023;64(8):761.

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

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Abstract

Purpose : The short wavelength sensitive (S) opsin genes (OPN1SW) of a wide variety of mammals have accumulated mutations that render them devoid of S cones. This alteration reduces these species to have a single cone type making them color-blind. Different OPN1SW mutations have been identified in each of several species (many of them nocturnal), indicating that tritanopia has arisen independently many times in mammalian evolution. The mutation in the Syrian hamster OPN1SW gene was repaired by gene editing in an attempt to restore S cone function in a strongly nocturnal rodent.

Methods : A single base pair deletion was corrected by pronuclear injection of the CRISPR-Cas9 complex into the zygotes of the hamster (Miao et al. 2018, JoVE 131 e56263) at Utah State University, and animals homozygous for the corrected gene were identified. Retinas were analyzed for OPN1SW mRNA, and also examined for S opsin protein in flat-mounts and cryosections using immunohistochemistry with antibodies against S opsin.

Results : Messenger RNA for the OPN1SW was found in homogenates of retinas from corrected animals. However, robust expression of S opsin protein was not detected using immunohistochemistry with a variety of antibodies targeting S opsin. A population of cones across the retina was found to have rounded, abnormal-appearing outer segments with faint S opsin signal.

Conclusions : Corrected S opsin DNA and mRNA were found in the corrected animal, demonstrating that single base pair deletions in opsin-encoding genes can be corrected in the germline using CRISPR-Cas9. However, S opsin protein was present in the hamster cones in small amounts at best. This suggests that hamsters may have further defects that interfere with the production of robust functional S cones.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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