The Lrat-/- Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa.

PURPOSE: We developed and phenotyped a pigmented knockout rat model for lecithin retinol acyltransferase (LRAT) using CRISPR/Cas9. The introduced mutation (c.12delA) is based on a patient group harboring a homologous homozygous frameshift mutation in the LRAT gene (c.12delC), causing a dysfunctional visual (retinoid) cycle.

METHODS

The introduced mutation was confirmed by DNA and RNA sequencing. The expression of Lrat was determined on both the RNA and protein level in wildtype and knockout animals using RT-PCR and immunohistochemistry. The retinal structure and function, as well as the visual behavior of the Lrat-/- and control rats, were characterized using scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), electroretinography (ERG) and vision-based behavioral assays.

RESULTS

Wildtype animals had high Lrat mRNA expression in multiple tissues, including the eye and liver. In contrast, hardly any expression was detected in Lrat-/- animals. LRAT protein was abundantly present in wildtype animals and absent in Lrat-/- animals. Lrat-/- animals showed progressively reduced ERG potentials compared to wildtype controls from two weeks of age onwards. Vison-based behavioral assays confirmed reduced vision. Structural abnormalities, such as overall retinal thinning, were observed in Lrat-/- animals. The retinal thickness in knockout rats was decreased to roughly 80% by four months of age. No functional or structural differences were observed between wildtype and heterozygote animals.

CONCLUSIONS

Our Lrat-/- rat is a new animal model for retinal dystrophy, especially for the LRAT-subtype of early-onset retinal dystrophies. This model has advantages over the existing mouse models and the RCS rat strain and can be used for translational studies of retinal dystrophies.