SummaryRetinitis pigmentosa and Leber congenital amaurosis are inherited retinal dystrophies that can be caused by mutations in the Crumbs homolog 1 (CRB1) gene. CRB1 is required for organizing apical-basal polarity and adhesion between photoreceptors and Müller glial cells.CRB1patient-derived induced pluripotent stem cells were differentiated intoCRB1retinal organoids that showed diminished expression of variant CRB1 protein observed by immunohistochemical analysis. Single-cell RNA sequencing revealed impact on, among others, the endosomal pathway and cell adhesion and migration inCRB1patient-derived retinal organoids compared with isogenic controls. Adeno-associated viral (AAV) vector-mediated hCRB2or hCRB1gene augmentation in Müller glial and photoreceptor cells partially restored the histological phenotype and transcriptomic profile ofCRB1patient-derived retinal organoids. Altogether, we show proof-of-concept that AAV.hCRB1or AAV.hCRB2treatment improved the phenotype ofCRB1patient-derived retinal organoids, providing essential information for future gene therapy approaches for patients with mutations in theCRB1gene.