Rhodopsin, the prototypical class-A G-protein coupled receptor, is a highly sensitive receptor for light that enables phototransduction in rod photoreceptors. Rhodopsin plays not only a sensory role but also a structural role as a major component of the rod outer segment disc, comprising over 90% of the protein content of the disc membrane. Mutations in RHO which lead to structural or functional abnormalities, including the autosomal recessive E150K mutation, result in rod dysfunction and death. Therefore, correction of deleterious rhodopsin mutations could rescue inherited retinal degeneration, as demonstrated for other visual genes such as RPE65 and PDE6B. In this study, we describe a CRISPR/Cas9 adenine base editing strategy to correct the E150K mutation and demonstrate precise in vivo editing in a Rho -E150K mouse model of autosomal recessive retinitis pigmentosa (RP). Using ultraviolet-visible spectroscopy, mass spectrometry, and the G-protein activation assay, we characterized wild-type rhodopsin and rhodopsin variants containing bystander base edits. Subretinal injection of dual-adeno-associated viruses delivering our base editing strategy yielded up to 44% Rho correction in homozygous Rho -E150K mice. Injection at postnatal day 15, but not later time points, restored rhodopsin expression, partially rescued retinal function, and partially preserved retinal structure. These findings demonstrate that in vivo base editing can restore the function of mutated structural and functional proteins in animal models of disease, including rhodopsin-associated RP and suggest that the timing of gene-editing is a crucial determinant of successful treatment outcomes for degenerative genetic diseases.

中文翻译：

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体内光感受器碱基编辑改善小鼠视紫红质-E150K 常染色体隐性遗传性视网膜色素变性


视紫红质是典型的 A 类 G 蛋白偶联受体，是一种高度敏感的光受体，可在杆状光感受器中进行光转导。视紫红质不仅起感觉作用，而且作为杆外段盘的主要成分也起结构作用，占盘膜蛋白质含量的 90% 以上。导致结构或功能异常的 RHO 突变，包括常染色体隐性遗传 E150K 突变，导致视杆功能障碍和死亡。因此，纠正有害的视紫红质突变可以挽救遗传性视网膜变性，如 RPE65 和 PDE6B 等其他视觉基因所证明的那样。在这项研究中，我们描述了一种 CRISPR/Cas9 腺嘌呤碱基编辑策略，以纠正 E150K 突变，并在常染色体隐性遗传性视网膜色素变性 （RP） 的 Rho -E150K 小鼠模型中展示了精确的体内编辑。使用紫外-可见光谱、质谱和 G 蛋白活化测定，我们表征了野生型视紫红质和包含旁观者碱基编辑的视紫红质变体。提供我们的碱基编辑策略的双腺相关病毒的视网膜下注射在纯合 Rho -E150K 小鼠中产生了高达 44% 的 Rho 校正。在出生后第 15 天注射，但不是更晚的时间点注射，恢复视紫红质表达，部分挽救视网膜功能，部分保留视网膜结构。这些发现表明，体内碱基编辑可以恢复疾病动物模型中突变结构和功能蛋白的功能，包括视紫红质相关 RP，并表明基因编辑的时机是退行性遗传病治疗结果成功的关键决定因素。