International Journal of Molecular Sciences Article The Lrat−/− Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa Céline Koster 1 , Koen T. van den Hurk 1, Colby F. Lewallen 2, Mays Talib 3, Jacoline B. ten Brink 1 , Camiel J. F. Boon 3,4 and Arthur A. Bergen 1,4,5,* 1 Department of Human Genetics Amsterdam, Section of Ophthalmogenetics, Amsterdam University Medical Centers (AUMC), University of Amsterdam (UvA), Location Meibergdreef, 1105 AZ Amsterdam, The Netherlands; [email protected] (C.K.); [email protected] (K.T.v.d.H.); [email protected] (J.B.t.B.) 2 Georgia Institute of Technology, G.W. Woodruff School of Mechanical Engineering, Atlanta, GA 30313, USA; [email protected] 3 Department of Ophthalmology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands; [email protected] (M.T.); [email protected] (C.J.F.B.) 4 Department of Ophthalmology, Amsterdam University Medical Centers (AUMC), University of Amsterdam (UvA), Location Meibergdreef, 1105 AZ Amsterdam, The Netherlands 5 The Netherlands Institute for Neuroscience (NIN-KNAW), 1105 BA Amsterdam, The Netherlands * Correspondence: [email protected] 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), Citation: Koster, C.; van den Hurk, causing a dysfunctional visual (retinoid) cycle. Methods: The introduced mutation was confirmed by K.T.; Lewallen, C.F.; Talib, M.; ten DNA and RNA sequencing. The expression of Lrat was determined on both the RNA and protein Brink, J.B.; Boon, C.J.F.; Bergen, A.A. level in wildtype and knockout animals using RT-PCR and immunohistochemistry. The retinal struc- The Lrat−/− Rat: CRISPR/Cas9 ture and function, as well as the visual behavior of the Lrat−/− and control rats, were characterized Construction and Phenotyping of a using scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), electroretinog- New Animal Model for Retinitis raphy (ERG) and vision-based behavioral assays. Results: Wildtype animals had high Lrat mRNA Pigmentosa. Int. J. Mol. Sci. 2021, 22, 7234. https://doi.org/10.3390/ expression in multiple tissues, including the eye and liver. In contrast, hardly any expression was −/− ijms22137234 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 Academic Editor: Stephanie wildtype controls from two weeks of age onwards. Vison-based behavioral assays confirmed re- C. Joachim duced 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 Received: 28 May 2021 age. No functional or structural differences were observed between wildtype and heterozygote Accepted: 29 June 2021 animals. Conclusions: Our Lrat−/− rat is a new animal model for retinal dystrophy, especially for Published: 5 July 2021 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. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: lecithin retinol acyltransferase (LRAT); pigmented rat model; retinal pigment epithelium published maps and institutional affil- (RPE); retinal degeneration; retinal dystrophy; retinitis pigmentosa iations. 1. Introduction Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and retinitis punctata This article is an open access article albescens (RPA) are severe early-onset retinal dystrophies that cause visual impairment, distributed under the terms and nystagmus, progressive nyctalopia, and finally, blindness. This heterogeneous retinal conditions of the Creative Commons dystrophy disease group is characterized by damage to the retinal pigment epithelium Attribution (CC BY) license (https:// (RPE)–photoreceptor (PR) complex. This results usually in progressive dysfunction of creativecommons.org/licenses/by/ the rod photoreceptor cells, often followed by progressive cone degeneration. RP, LCA, 4.0/). and RPA are caused by mutations in virtually all genes encoding proteins acting in the Int. J. Mol. Sci. 2021, 22, 7234. https://doi.org/10.3390/ijms22137234 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 7234 2 of 22 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 22 retinoid cycle [1–4]. Indeed, for normal vision, a functionally valid retinoid cycle is es- sential:cycle [1–4]. In the Indeed, healthy for situation,normal vision, vitamin a functionally A (retinol) valid is the retinoid primary cycle substrate is essential: for In several the functionalhealthy situation, retinoids’ vitamin biosynthesis A (retinol) in the is retinoidthe primary cycle. substrate Then, the for vitamin several A-derivatives functional areretinoids’ shuttled biosynthesis from the RPE in the to retinoid the PRs. cycle. There, Then, opsins the vitamin are light-activated A‐derivatives andare shuttled the visual pigmentsfrom the RPE transform to the PRs. the lightThere, energy opsins inare a light cellular‐activated signal, and initiating the visual the pigments visual transform cascade and resultingthe light in energy a physiological in a cellular response signal, in initiating the PR cell. the Aftervisual light cascade activation, and resulting the cycle in regen- a eratesphysiological the visual response pigments in the that PR are cell. used After after light light activation, activation the cycle of rhodopsin regenerates(see the Figurevisual 1). Uponpigments photoactivation, that are used a configurationalafter light activation change of of rhodopsin the visual pigment(see Figure 11- cis1). -retinalUpon to all-photoactivation,trans-retinal is a induced configurational in the PR change cells’ of outer the visual segments. pigment Subsequently, 11‐cis‐retinal all- to transall‐trans-retinal‐ isretinal reduced is induced to all-trans in the-retinol PR cells’ and outer diffuses segments. from Subsequently, the PRs back all to‐trans to the‐retinal RPE is cells. reduced In the RPE,to all all-‐transtrans‐retinol-retinol and is diffuses esterified from to the all- PRstrans back-retinyl-ester to to the RPE by cells. the enzyme In the RPE, lecithin:retinol all‐trans‐ acetyltransferaseretinol is esterified (LRAT), to all‐trans after‐retinyl which‐ester all- transby the-retinyl-ester enzyme lecithin:retinol is subsequently acetyltransferase the substrate (LRAT), after which all‐trans‐retinyl‐ester is subsequently the substrate for the enzyme for the enzyme retinal pigment epithelium-specific protein 65 kDa (RPE65). RPE65 converts retinal pigment epithelium‐specific protein 65 kDa (RPE65). RPE65 converts all‐trans‐ all-trans-retinyl-ester to 11-cis-retinol, after which 11-cis-retinol is oxidized by retinol dehy- retinyl‐ester to 11‐cis‐retinol, after which 11‐cis‐retinol is oxidized by retinol dehydrogenase drogenase (RDH) enzymes to 11-cis-retinal. Finally, to complete the cycle, 11-cis-retinal is (RDH) enzymes to 11‐cis‐retinal. Finally, to complete the cycle, 11‐cis‐retinal is shuttled back shuttled back to the PRs, where it can be used for a new round of phototransduction. to the PRs, where it can be used for a new round of phototransduction. FigureFigure 1. A 1. schematic A schematic overview overview of of the the visualvisual cycle in in the the photoreceptors photoreceptors (PRs) (PRs) and and the theretinal retinal pigment pigment epithelium epithelium (RPE). (RPE). In In thethe PRs,PRs, 11 11-‐ciscis‐retinal-retinal couples couples to an to opsin an opsin protein, protein, forming forming rhodopsin. rhodopsin. Upon activation Upon by activation photons, by 11‐cis photons,‐retinal is 11- isomerizedcis-retinal is to all‐trans‐retinal. The retinol dehydrogenases (encoded by RDH8, RDH12, RDH14) reduce all‐trans‐retinal to all‐trans‐ isomerized to all-trans-retinal. The retinol dehydrogenases (encoded by RDH8, RDH12, RDH14) reduce all-trans-retinal to retinol, and this metabolite is moved to the RPE by retinoid‐binding protein (encoded by RBP3). In the RPE, it is esterified by all-translecithin:retinol-retinol, and acyltransferase this metabolite (encoded is moved by LRAT to the), after RPE which by retinoid-binding it is converted to 11 protein‐cis‐retinol (encoded by retinal by RBP3pigment). In epithelium the RPE,‐ it is esterifiedspecific by 65 lecithin:retinol kDa protein (encoded acyltransferase by RPE65 (encoded). Retinol by dehydrogenasesLRAT), after which (encoded it is by converted RDH5 and to 11-RDH11cis-retinol) convert by 11 retinal‐cis‐retinol pigment epithelium-specificto 11‐cis‐retinal, and 65 kDaretinoid protein‐binding (encoded protein by movesRPE65 it back). Retinol to the dehydrogenasesPR. For further explanation, (encoded bysee RDH5the text.and RDH11) convert 11-cis-retinol to 11-cis-retinal, and retinoid-binding protein moves it back to the PR. For further explanation, see the text. Thus, LRAT as well as RPE65 are essential for the regeneration of functional visual pigmentThus, in LRAT the part as well of the as retinoid RPE65 arecycle essential that takes for place