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

Céline Koster,Mays Talib,Jacoline Ten Brink,Koen Van Den Hurk,Camiel Boon,Colby Lewallen,Arthur Bergen

doi:10.3390/ijms22137234

Abstract

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.

Highlights

Retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and retinitis punctata albescens (RPA) are severe early-onset retinal dystrophies that cause visual impairment, nystagmus, progressive nyctalopia, and blindness. This heterogeneous retinal dystrophy disease group is characterized by damage to the retinal pigment epithelium (RPE)–photoreceptor (PR) complex
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We confirmed that the targeted mutation in both genomic DNA as cDNA in our Lrat−/− animals was the causative mutation for the in vivo vision-related phenotype that we observed in this strain

Summary

Introduction

Retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and retinitis punctata albescens (RPA) are severe early-onset retinal dystrophies that cause visual impairment, nystagmus, progressive nyctalopia, and blindness. (BCI):dLaRtaAbTasper,oAtecincession presence is confirmed in the liver of Lrat+/+ animals and (almost completely) absent in Lrat−/− liver tissue. (C): LRAT protein presence is confirmed in the liver of Lrat+/+ animals and (almost completely) absent in Lrat−/− liver tissue. In agreement with the mRNA expression, we observed, using immunofluorescence, that LRAT protein was abundantly present in liver sections of Lrat+/+ animals and (almost completely) absent in liver sections of Lrat−/− animals (Figure 4C). Wildtype control animals of various ages did not show any significant differences in ERG amplitudes between time points (n = 6; see Supplementary Figure S4). At 5 weeks of age, dark-adapted a-wave amplitudes were hardly recordable, and b-wave amplitudes were strongly decreased for Lrat−/− animals for all light intensities (Figure 8A,B).

Discussion

Construction of the Animal Model

Mutation Analysis and Expression of the Lrat Gene

LRAT Detection Using Immunofluorescence

Findings

Statistical Analyses Performed