Prominin 1 is crucial for the early development of photoreceptor outer segments.

Prominin 1 (PROM1) is a pentaspan transmembrane glycoprotein localized on the nascent photoreceptor discs. Mutations in PROM1 are linked to various retinal diseases. In this study, we assessed the role of PROM1 in photoreceptor biology and physiology using the PROM1 knockout murine model (rd19). Our study found that PROM1 is essential for vision and photoreceptor development. We found an early reduction in photoreceptor response beginning at post-natal day 12 (P12) before eye opening in the absence of PROM1 with no apparent loss in photoreceptor cells. However, at this stage, we observed an increased glial cell activation, indicative of cell damage. Contrary to our expectations, dark rearing did not mitigate photoreceptor degeneration or vision loss in PROM1 knockout mice. In addition to physiological defects seen in PROM1 knockout mice, ultrastructural analysis revealed malformed outer segments characterized by whorl-like continuous membranes instead of stacked disks. In parallel to the reduced rod response at P12, proteomics revealed a significant reduction in the levels of protocadherin, a known interactor of PROM1, and rod photoreceptor outer segment proteins, including rhodopsin. Overall, our results underscore the indispensable role of PROM1 in photoreceptor development and maintenance of healthy vision.

Dark continuous noise from mutant G90D-rhodopsin predominantly underlies congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is an inherited retinal disease that causes a profound loss of rod sensitivity without severe retinal degeneration. One well-studied rhodopsin point mutant, G90D-Rho, is thought to cause CSNB because of its constitutive activity in darkness causing rod desensitization. However, the nature of this constitutive activity and its precise molecular source have not been resolved for almost 30 y. In this study, we made a knock-in (KI) mouse line with a very low expression of G90D-Rho (equal in amount to ~0.1% of normal rhodopsin, WT-Rho, in WT rods), with the remaining WT-Rho replaced by REY-Rho, a mutant with a very low efficiency of activating transducin due to a charge reversal of the highly conserved ERY motif to REY. We observed two kinds of constitutive noise: one being spontaneous isomerization (R*) of G90D-Rho at a molecular rate (R* s-1) 175-fold higher than WT-Rho and the other being G90D-Rho-generated dark continuous noise comprising low-amplitude unitary events occurring at a very high molecular rate equivalent in effect to ~40,000-fold of R* s-1 from WT-Rho. Neither noise type originated from G90D-Opsin because exogenous 11-cis-retinal had no effect. Extrapolating the above observations at low (0.1%) expression of G90D-Rho to normal disease exhibited by a KI mouse model with RhoG90D/WTand RhoG90D/G90D genotypes predicts the disease condition very well quantitatively. Overall, the continuous noise from G90D-Rho therefore predominates, constituting the major equivalent background light causing rod desensitization in CSNB.

A subgroup of light-driven sodium pumps with an additional Schiff base counterion.

Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.

Tamoxifen protects photoreceptors in the sodium iodate model.

Because the selective estrogen receptor modulator tamoxifen was shown to be retina-protective in the light damage and rd10 models of retinal degeneration, the purpose of this study was to test whether tamoxifen is retina-protective in a model where retinal pigment epithelium (RPE) toxicity appears to be the primary insult: the sodium iodate (NaIO3) model. C57Bl/6J mice were given oral tamoxifen (in the diet) or the same diet lacking tamoxifen, then given an intraperitoneal injection of NaIO3 at 25 mg/kg. The mice were imaged a week later using optical coherence tomography (OCT). ImageJ with a custom macro was utilized to measure retinal thicknesses in OCT images. Electroretinography (ERG) was used to measure retinal function one week post-injection. After euthanasia, quantitative real-time PCR (qRT-PCR) was performed. Tamoxifen administration partially protected photoreceptors. There was less photoreceptor layer thinning in OCT images of tamoxifen-treated mice. qRT-PCR revealed, in the tamoxifen-treated group, less upregulation of antioxidant and complement factor 3 mRNAs, and less reduction in the rhodopsin and short-wave cone opsin mRNAs. Furthermore, ERG results demonstrated preservation of photoreceptor function for the tamoxifen-treated group. Cone function was better protected than rods. These results indicate that tamoxifen provided structural and functional protection to photoreceptors against NaIO3. RPE cells were not protected. These neuroprotective effects suggest that estrogen-receptor modulation may be retina-protective. The fact that cones are particularly protected is intriguing given their importance for human visual function and their survival until the late stages of retinitis pigmentosa. Further investigation of this protective pathway could lead to new photoreceptor-protective therapeutics.

Rhodopsin mislocalization drives ciliary dysregulation in a novel autosomal dominant retinitis pigmentosa knock-in mouse model.

Rhodopsin mislocalization encompasses various blind conditions. Rhodopsin mislocalization is the primary factor leading to rod photoreceptor dysfunction and degeneration in autosomal dominant retinitis pigmentosa (adRP) caused by class I mutations. In this study, we report a new knock-in mouse model that harbors a class I Q344X mutation in the endogenous rhodopsin gene, which causes rod photoreceptor degeneration in an autosomal dominant pattern. In RhoQ344X/+ mice, mRNA transcripts from the wild-type (Rho) and RhoQ344X mutant rhodopsin alleles are expressed at equal levels. However, the amount of RHOQ344X mutant protein is 2.7 times lower than that of wild-type rhodopsin, a finding consistent with the rapid degradation of the mutant protein. Immunofluorescence microscopy indicates that RHOQ344X is mislocalized to the inner segment and outer nuclear layers of rod photoreceptors in both RhoQ344X/+ and RhoQ344X/Q344X mice, confirming the essential role of the C-terminal VxPx motif in promoting OS delivery of rhodopsin. The mislocalization of RHOQ344X is associated with the concurrent mislocalization of wild-type rhodopsin in RhoQ344X/+ mice. To understand the global changes in proteostasis, we conducted quantitative proteomics analysis and found attenuated expression of rod-specific OS membrane proteins accompanying reduced expression of ciliopathy causative gene products, including constituents of BBSome and axonemal dynein subunit. Those studies unveil a novel negative feedback regulation involving ciliopathy-associated proteins. In this process, a defect in the trafficking signal leads to a reduced quantity of the trafficking apparatus, culminating in a widespread reduction in the transport of ciliary proteins.

The Logistical Backbone of Photoreceptor Cell Function: Complementary Mechanisms of Dietary Vitamin A Receptors and Rhodopsin Transporters.

In this review, we outline our current understanding of the mechanisms involved in the absorption, storage, and transport of dietary vitamin A to the eye, and the trafficking of rhodopsin protein to the photoreceptor outer segments, which encompasses the logistical backbone required for photoreceptor cell function. Two key mechanisms of this process are emphasized in this manuscript: ocular and systemic vitamin A membrane transporters, and rhodopsin transporters. Understanding the complementary mechanisms responsible for the generation and proper transport of the retinylidene protein to the photoreceptor outer segment will eventually shed light on the importance of genes encoded by these proteins, and their relationship on normal visual function and in the pathophysiology of retinal degenerative diseases.

Disease modeling and pharmacological rescue of autosomal dominant retinitis pigmentosa associated with RHO copy number variation.

Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder, causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene (RHO) account for ~25% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first-ever reported mono-allelic copy number variation (CNV) in RHO as a novel cause of adRP. We (a) show advanced retinal degeneration in a male patient (68 years of age) harboring four transcriptionally active intact copies of rhodopsin, (b) recapitulated the clinical phenotypes using retinal organoids, and (c) assessed the utilization of a small molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patients associated with RHO-CNV. Patient retinal organoids showed photoreceptors dysgenesis, with rod photoreceptors displaying stunted outer segments with occasional elongated cilia-like projections (microscopy); increased RHO mRNA expression (quantitative real-time PCR [qRT-PCR] and bulk RNA sequencing); and elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300 days) culture time period when compared against control organoids. Lastly, we utilized PR3 to target NR2E3, an upstream regulator of RHO, to alter RHO expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provide a proof-of-principle for personalized medicine and suggest that RHO expression requires precise control. Taken together, this study supports the clinical data indicating that RHO-CNV associated adRPdevelops as a result of protein overexpression, thereby overloading the photoreceptor post-translational modification machinery.

Modeling autosomal dominant retinitis pigmentosa by using patient-specific retinal organoids with a class-3 RHO mutation.

Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RHO-adRP) causes progressive vision loss and is potentially incurable, accounting for 25% of adRP cases. Studies on RHO-adRP mechanism were at large based on the biochemical and cellular properties, especially class-3. Nonetheless, the absence of an appropriate model for class-3 RHO-adRP has impeded comprehensive exploration. Here, induced pluripotent stem cells (iPSCs) were generated from a healthy control and two sibling RP patients with the same point mutation, c.403C>T (p.R135W). The first three-dimensional (3D) retinal organoid model of a class-3 RHO point mutation from patient-derived iPSCs was generated. Significant defects were observed in rod photoreceptors in terms of localization, morphology, transcriptional profiling and single cell resolution, to better understand the human disease resulting from RHO mutations from a developmental perspective. This first human model of class-3 RHO-adRP provides a representation of patient's retina in vitro and displays features of RHO-adRP retinal organoids relevant for therapeutic development.

Multiple retinal isomerizations during the early phase of the bestrhodopsin photoreaction.

Bestrhodopsins constitute a class of light-regulated pentameric ion channels that consist of one or two rhodopsins in tandem fused with bestrophin ion channel domains. Here, we report on the isomerization dynamics in the rhodopsin tandem domains of Phaeocystis antarctica bestrhodopsin, which binds all-trans retinal Schiff-base (RSB) absorbing at 661 nm and, upon illumination, converts to the meta-stable P540 state with an unusual 11-cis RSB. The primary photoproduct P682 corresponds to a mixture of highly distorted 11-cis and 13-cis RSB directly formed from the excited state in 1.4 ps. P673 evolves from P682 in 500 ps and contains highly distorted 13-cis RSB, indicating that the 11-cis fraction in P682 converts to 13-cis. Next, P673 establishes an equilibrium with P595 in 1.2 µs, during which RSB converts to 11-cis and then further proceeds to P560 in 48 µs and P540 in 1.0 ms while remaining 11-cis. Hence, extensive isomeric switching occurs on the early ground state potential energy surface (PES) on the hundreds of ps to µs timescale before finally settling on a metastable 11-cis photoproduct. We propose that P682 and P673 are trapped high up on the ground-state PES after passing through either of two closely located conical intersections that result in 11-cis and 13-cis RSB. Co-rotation of C11=C12 and C13=C14 bonds results in a constricted conformational landscape that allows thermal switching between 11-cis and 13-cis species of highly strained RSB chromophores. Protein relaxation may release RSB strain, allowing it to evolve to a stable 11-cis isomeric configuration in microseconds.

Picosecond quantum-classical dynamics reveals that the coexistence of light-induced microbial and animal chromophore rotary motion modulates the isomerization quantum yield of heliorhodopsin.

Rhodopsins are light-responsive proteins forming two vast and evolutionary distinct superfamilies whose functions are invariably triggered by the photoisomerization of a single retinal chromophore. In 2018 a third widespread superfamily of rhodopsins called heliorhodopsins was discovered using functional metagenomics. Heliorhodopsins, with their markedly different structural features with respect to the animal and microbial superfamilies, offer an opportunity to study how evolution has manipulated the chromophore photoisomerization to achieve adaptation. One question is related to the mechanism of such a reaction and how it differs from that of animal and microbial rhodopsins. To address this question, we use hundreds of quantum-classical trajectories to simulate the spectroscopically documented picosecond light-induced dynamics of a heliorhodopsin from the archaea thermoplasmatales archaeon (TaHeR). We show that, consistently with the observations, the trajectories reveal two excited state decay channels. However, inconsistently with previous hypotheses, only one channel is associated with the -C13C14- rotation of microbial rhodopsins while the second channel is characterized by the -C11C12- rotation typical of animal rhodopsins. The fact that such -C11C12- rotation is aborted upon decay and ground state relaxation, explains why illumination of TaHeR only produces the 13-cis isomer with a low quantum efficiency. We argue that the documented lack of regioselectivity in double-bond excited state twisting motion is the result of an "adaptation" that could be completely lost via specific residue substitutions modulating the steric hindrance experienced along the isomerization motion.

Light regulation of rhodopsin distribution during outer segment renewal in murine rod photoreceptors.

Vision under dim light relies on primary cilia elaborated by rod photoreceptors in the retina. This specialized sensory structure, called the rod outer segment (ROS), comprises hundreds of stacked, membranous discs containing the light-sensitive protein rhodopsin, and the incorporation of new discs into the ROS is essential for maintaining the rod's health and function. ROS renewal appears to be primarily regulated by extrinsic factors (light); however, results vary depending on different model organisms. We generated two independent transgenic mouse lines where rhodopsin's fate is tracked by a fluorescently labeled rhodopsin fusion protein (Rho-Timer) and show that rhodopsin incorporation into nascent ROS discs appears to be regulated by both external lighting cues and autonomous retinal clocks. Live-cell imaging of the ROS isolated from mice exposed to six unique lighting conditions demonstrates that ROS formation occurs in a periodic manner in cyclic light, constant darkness, and artificial light/dark cycles. This alternating bright/weak banding of Rho-Timer along the length of the ROS relates to inhomogeneities in rhodopsin density and potential points of structural weakness. In addition, we reveal that prolonged dim ambient light exposure impacts not only the rhodopsin content of new discs but also that of older discs, suggesting a dynamic interchange of material between new and old discs. Furthermore, we show that rhodopsin incorporation into the ROS is greatly altered in two autosomal recessive retinitis pigmentosa mouse models, potentially contributing to the pathogenesis. Our findings provide insights into how extrinsic (light) and intrinsic (retinal clocks and genetic mutation) factors dynamically regulate mammalian ROS renewal.

Glu1022.53-Mediated Early Conformational Changes in the Process of Light-Induced Green Cone Pigment Activation.

Ligand-triggered activation of G protein-coupled receptors (GPCRs) relies on the phenomenon of loose allosteric coupling, which involves conformational alterations spanning from the extracellular ligand-binding domain to the cytoplasmic region, where interactions with G proteins occur. During the GPCR activation process, several intermediate and equilibrium states orchestrate the movement of the flexible and rigid transmembrane (TM) segments of the GPCR. Monitoring early conformational changes is important in unraveling the structural intricacies of the loose allosteric coupling. Here, we focus on the lumi intermediate formed by thermal relaxation from the initial photointermediate, batho in primate green cone pigment (MG), a light-sensitive GPCR responsible for color vision. Our findings from light-induced Fourier transform infrared difference spectroscopy reveal its similarity with rhodopsin, which mediates twilight vision, specifically involving the flip motion of the ß-ionone ring, the relaxation of the torsional structure of the retinal, and local perturbations in the α-helix upon lumi intermediate formation. Conversely, we observe a hydrogen bond modification specific to MG's protonated carboxylic acid, identifying its origin as Glu1022.53 situated in TM2. The weakening of the hydrogen bond strength at Glu1022.53 during the transition from the batho to the lumi intermediates corresponds to a slight outward movement of TM2. Additionally, within the X-ray crystal structure of the rhodopsin lumi intermediate, we note the relocation of the Met862.53 side chain in TM2, expanding the volume of the retinal binding pocket. Consequently, the position of 2.53 emerges as the early step in the conformational shift toward light-induced activation. Moreover, given the prevalence of IR-insensitive hydrophobic amino acids at position 2.53 in many rhodopsin-like GPCRs, including rhodopsin, the hydrogen bond alteration in the C═O stretching band at Glu1022.53 of MG can be used as a probe for tracing conformational changes during the GPCR activation process.

Color Tuning in Bovine Rhodopsin through Polarizable Embedding.

Bovine rhodopsin is among the most studied proteins in the rhodopsin family. Its primary activation mechanism is the photoisomerization of 11-cis retinal, triggered by the absorption of a UV-visible photon. Different mutants of the same rhodopsin show different absorption wavelengths due to the influence of the specific amino acid residues forming the cavity in which the retinal chromophore is embedded, and rhodopsins activated at different wavelengths are, for example, exploited in the field of optogenetics. In this letter, we present a procedure for systematically investigating color tuning in models of bovine rhodopsin and a set of its mutants embedded in a membrane bilayer. Vertical excitation energy calculations were carried out with the polarizable embedding potential for describing the environment surrounding the chromophore. We show that polarizable embedding outperformed regular electrostatic embedding in determining both the vertical excitation energies and associated oscillator strengths of the systems studied.

Accurate Detection of Convergent Mutations in Large Protein Alignments With ConDor.

Evolutionary convergences are observed at all levels, from phenotype to DNA and protein sequences, and changes at these different levels tend to be correlated. Notably, convergent mutations can lead to convergent changes in phenotype, such as changes in metabolism, drug resistance, and other adaptations to changing environments. We propose a two-component approach to detect mutations subject to convergent evolution in protein alignments. The "Emergence" component selects mutations that emerge more often than expected, while the "Correlation" component selects mutations that correlate with the convergent phenotype under study. With regard to Emergence, a phylogeny deduced from the alignment is provided by the user and is used to simulate the evolution of each alignment position. These simulations allow us to estimate the expected number of mutations in a neutral model, which is compared to the observed number of mutations in the data studied. In Correlation, a comparative phylogenetic approach, is used to measure whether the presence of each of the observed mutations is correlated with the convergent phenotype. Each component can be used on its own, for example Emergence when no phenotype is available. Our method is implemented in a standalone workflow and a webserver, called ConDor. We evaluate the properties of ConDor using simulated data, and we apply it to three real datasets: sedge PEPC proteins, HIV reverse transcriptase, and fish rhodopsin. The results show that the two components of ConDor complement each other, with an overall accuracy that compares favorably to other available tools, especially on large datasets.

Chromophore-Protein Interactions Affecting the Polyene Twist and π-π* Energy Gap of the Retinal Chromophore in Schizorhodopsins.

The properties of a prosthetic group are broadened by interactions with its neighboring residues in proteins. The retinal chromophore in rhodopsins absorbs light, undergoes structural changes, and drives functionally important structural changes in proteins during the photocycle. It is therefore crucial to understand how chromophore-protein interactions regulate the molecular structure and electronic state of chromophores in rhodopsins. Schizorhodopsin is a newly discovered subfamily of rhodopsins found in the genomes of Asgard archaea, which are extant prokaryotes closest to the last common ancestor of eukaryotes and of other microbial species. Here, we report the effects of a hydrogen bond between a retinal Schiff base and its counterion on the twist of the polyene chain and the color of the retinal chromophore. Correlations between spectral features revealed the unexpected fact that the twist of the polyene chain is reduced as the hydrogen bond becomes stronger, suggesting that the twist is caused by tight atomic contacts between the chromophore and nearby residues. In addition, the strength of the hydrogen bond is the primary factor affecting the color-tuning of the retinal chromophore in schizorhodopsins. The findings of this study are valuable for manipulating the molecular structure and electronic state of the chromophore by controlling chromophore-protein interactions.

Deciphering the role of glycosaminoglycans in GPCR signaling.

G protein-coupled receptors (GPCR) and glycosaminoglycans (GAGs) are two essential components of the cell surface that regulate physiological processes in the body. GPCRs are the most extensive family of transmembrane receptors that control cellular responses to extracellular stimuli, while GAGs are polysaccharides that contribute to the function of the extracellular matrix (ECM). Due to their proximity to the plasma membrane, GAGs participate in signal transduction by interacting with various extracellular molecules and cell surface receptors. GAGs can directly interact with certain GPCRs or their ligands (chemokines, peptide hormones and neuropeptides, structural proteins, and enzymes) from the glutamate receptor family, the rhodopsin receptor family, the adhesion receptor family, and the secretin receptor family. These interactions have recently become an emerging topic, providing a new avenue for understanding how GPCR signaling is regulated. This review discusses our current state of knowledge about the role of GAGs in GPCR signaling and function.

Convergent mechanism underlying the acquisition of vertebrate scotopic vision.

High sensitivity of scotopic vision (vision in dim light conditions) is achieved by the rods' low background noise, which is attributed to a much lower thermal activation rate (kth) of rhodopsin compared with cone pigments. Frogs and nocturnal geckos uniquely possess atypical rods containing noncanonical cone pigments that exhibit low kth, mimicking rhodopsin. Here, we investigated the convergent mechanism underlying the low kth of rhodopsins and noncanonical cone pigments. Our biochemical analysis revealed that the kth of canonical cone pigments depends on their absorption maximum (λmax). However, rhodopsin and noncanonical cone pigments showed a substantially lower kth than predicted from the λmax dependency. Given that the λmax is inversely proportional to the activation energy of the pigments in the Hinshelwood distribution-based model, our findings suggest that rhodopsin and noncanonical cone pigments have convergently acquired low frequency of spontaneous-activation attempts, including thermal fluctuations of the protein moiety, in the molecular evolutionary processes from canonical cone pigments, which contributes to highly sensitive scotopic vision.

Voluntary exercise preserves visual function and reduces inflammatory response in an adult mouse model of autosomal dominant retinitis pigmentosa.

Whole-body physical exercise has been shown to promote retinal structure and function preservation in animal models of retinal degeneration. It is currently unknown how exercise modulates retinal inflammatory responses. In this study, we investigated cytokine alterations associated with retinal neuroprotection induced by voluntary running wheel exercise in a retinal degeneration mouse model of class B1 autosomal dominant retinitis pigmentosa, I307N Rho. I307N Rho mice undergo rod photoreceptor degeneration when exposed to bright light (induced). Our data show, active induced mice exhibited significant preservation of retinal and visual function compared to inactive induced mice after 4 weeks of exercise. Retinal cytokine expression revealed significant reductions of proinflammatory chemokines, keratinocyte-derived chemokine (KC) and interferon gamma inducible protein-10 (IP-10) expression in active groups compared to inactive groups. Through immunofluorescence, we found KC and IP-10 labeling localized to retinal vasculature marker, collagen IV. These data show that whole-body exercise lowers specific retinal cytokine expression associated with retinal vasculature. Future studies should determine whether suppression of inflammatory responses is requisite for exercise-induced retinal protection.

Compensation of inner retina to early-stage photoreceptor degeneration in a RhoP23H/+ mouse model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is an inherited retinal disorder characterized by the degeneration of photoreceptors. RhoP23H/+ mice, which carry a Pro23His mutation in the RHODOPSIN (Rho) gene, are one of the most studied animal models for RP. However, except for the photoreceptors, other retinal neural cells have not been fully investigated in this model. Here, we record the temporal changes of the retina by optical coherence tomography (OCT) imaging of the RhoP23H/+ mice, from early to mid-phase of retinal degeneration. Based on thickness analysis, we identified a natural retinal thickness adaption in wild-type mice during early adulthood and observed morphological compensation of the inner retina layer to photoreceptor degeneration in the RhoP23H/+ mice, primarily on the inner nuclear layer (INL). RhoP23H/+ mice findings were further validated via: histology showing the negative correlation of INL and ONL thicknesses; as well as electroretinogram (ERG) showing an increased b-wave to a-wave ratio. These results unravel the sequential morphologic events in this model and suggest a better understanding of retinal degeneration of RP for future studies.

Knockout and Replacement Gene Surgery to Treat Rhodopsin-Mediated Autosomal Dominant Retinitis Pigmentosa.

Mutations in the rhodopsin (RHO) gene are the predominant causes of autosomal dominant retinitis pigmentosa (adRP). Given the diverse gain-of-function mutations, therapeutic strategies targeting specific sequences face significant challenges. Here, we provide a universal approach to conquer this problem: we have devised a CRISPR-Cas12i-based, mutation-independent gene knockout and replacement compound therapy carried by a dual AAV2/8 system. In this study, we successfully delayed the progression of retinal degeneration in the classic mouse disease model RhoP23H, and also RhoP347S, a new native mouse mutation model we developed. Our research expands the horizon of potential options for future treatments of RHO-mediated adRP.