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The mouse and human retina contain three major Crumbs homologue-1 (CRB1) isoforms. CRB1-A and CRB1-B have cell-type-specific expression patterns making the choice of gene augmentation strategy unclear. Gene editing may be a viable alternative for the amelioration of CRB1-associated retinal degenerations. To assess the prevalence and spectrum of CRB1-associated pathogenic variants amenable to base and prime editing, we carried out an analysis of the Leiden Open Variation Database. Editable variants accounted for 54.5% for base editing and 99.8% for prime editing of all CRB1 pathogenic variants in the Leiden Open Variation Database. The 10 most common editable pathogenic variants for CRB1 accounted for 34.95% of all pathogenic variants, with the c.2843G>A, p.(Cys948Tyr) being the most common editable CRB1 variant. These findings outline the next step toward developing base and prime editing therapeutics as an alternative to gene augmentation for the amelioration of CRB1-associated retinal degenerations., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Mutations in the Crumbs-homologue-1 (CRB1) gene lead to a spectrum of severe inherited retinal diseases, including retinitis pigmentosa (RP). The establishment of a genotype-phenotype correlation in CRB1 patients has been difficult due to the substantial variability and phenotypic overlap between CRB1-associated diseases. This phenotypic modulation may be due to several factors, including genetic modifiers, deep intronic mutations, isoform diversity, and copy number variations. Induced pluripotent stem cell (iPSC)-derived patient retinal organoids are novel tools that can provide sensitive, quantitative, and scalable phenotypic assays. CRB1 RP patient iPSC-derived retinal organoids have shown reproducible phenotypes compared to healthy retinal organoids. However, having genetically defined iPSC isogenic controls that take into account potential phenotypic modulation is crucial. In this study, we generated iPSC from an early-onset CRB1 patient and developed a correction strategy for the c.2480G>T, p.(Gly827Val) CRB1 mutation using CRISPR/Cas9-mediated homology-directed repair., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Mutations in peripherin 2 (PRPH2) are associated with a spectrum of inherited retinal diseases (IRDs) including retinitis pigmentosa (RP) and macular degeneration. As PRPH2 is localized to cone and rod outer segments, mutations in PRPH2 lead the disorganization or absence of photoreceptor outer segments. Here, we report on a patient with PRPH2-linked RP who exhibited widespread RPE atrophy with a central area of macular atrophy sparing the fovea. In future studies, we plan to model the pathobiology of PRPH2-based RP using induced pluripotent stem cell (iPSC)-derived retinal organoids. To effectively model rare mutations using iPSC-derived retinal organoids, we first require a strategy that can install the desired mutation in healthy wild-type iPSC, which can efficiently generate well-laminated retinal organoids. In this study, we developed an efficient prime editing strategy for the installation of the pathogenic PRPH2 c.828+1 G>A splice-site mutation underlying our patient's disease., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Prime editing (PE) is a novel, double-strand break (DSB)-independent gene editing technology that represents an exciting avenue for the treatment of inherited retinal diseases (IRDs). Given the extensive and heterogenous nature of the 280 genes associated with IRDs, genome editing has presented countless complications. However, recent advances in genome editing technologies have identified PE to have tremendous potential, with the capability to ameliorate small deletions and insertions in addition to all twelve possible transition and transversion mutations. The current PE system is based on the fusion of the Streptococcus pyogenes Cas9 (SpCas9) nickase H840A mutant and an optimized Moloney murine leukemia virus (MMLV) reverse-transcriptase (RT) in conjunction with a PE guide RNA (pegRNA). In this study, we developed a prime editor based on the avian myeloblastosis virus (AMV)-RT and showed its applicability for the installation of the PRPH2 c.828+1G>A mutation in HEK293 cells., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)