Successful generation of a gene-edited 13-lined ground squirrel with an abnormal retinal phenotype

Abstract

Purpose : To generate a germline transgenic 13-lined ground squirrel (13-LGS) with an abnormal retinal phenotype that models a human condition.

Methods : Gene-edited 13-LGS were generated using the embryo microinjection method. Pronuclear or 2-cell stage embryos were collected from euthanized female 13-LGS one day after natural mating. CRISPR/Cas9 solution (3μg/ml) targeting the tyrosinase gene (Tyr) was microinjected into each nucleus. One hour after injection, embryos were surgically transferred into the oviducts of pseudo-pregnant recipient females. At weaning of resulting pups, genomic DNA was extracted from tail and ear tissues and any mutations were identified by Sanger sequencing. Both retinas of one mutant pup (age 6 mo) were imaged non-invasively with scanning light ophthalmoscopy (SLO) and optical coherence tomography (OCT) to assess the retinal phenotype. Measurements of choroidal thickness and total, inner, and outer retinal thickness were made on single, raw OCT vertical B-scans from 16.5° inferior of the optic nerve head (ONH) to 3° superior of the ONH.

Results : Two pups (1 male and 1 female) with mutations of the Tyr gene were generated. The male’s genotype showed a mosaic with low levels of indel signals. The female’s genotype showed a heterozygous 1bp insertion. SLO images of her retinas demonstrated bilateral abnormal reflectance patterns consistent with altered retinal pigmentation. These SLO abnormalities were also visible in en face summed volume projection images from the OCT volumes. Thickness values for all retinal layers were within the normative range (derived from a group of 1-year-old animals: 16M, 16F; n = 36 eyes).

Conclusions : This report demonstrates that live offspring from gene-edited 13-LGS embryos are indeed achievable and may harbor an abnormal ocular phenotype. Germline transmission of the prospective female founder’s mutation will be examined at sexual maturity, after she completes her first hibernation. We will continue to refine our transgenesis methods and expand our target genes as we explore the utility of this diurnal rodent model of human vision.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.