Retinal organoids mirror CRISPR-Cas9 gene editing efficiency observed in vivo.

Human retinal organoids are in vitro 3D structures that recapitulate key molecular and structural characteristics of the in vivo retina. They include all essential retinal cell types including photoreceptors, making them relevant models for preclinical development of gene therapies. A critical knowledge gap exists in understanding their utility for gene editing therapy optimization. We assessed the potential of retinal organoids for optimizing CRISPR-Cas9-mediated gene editing, focusing on the therapeutically relevant RHO gene implicated in autosomal dominant retinitis pigmentosa (adRP). Using retinal organoids, in vitro HEK293T cells, and two humanized mouse models carrying different RHO mutations, we compared editing efficiencies. We observed that retinal organoids have lower transfection efficiency compared to HEK293T cells. Notably, they exhibited editing efficiencies more closely aligned with those found in vivo. We also observed similar delivery patterns of CRISPR-Cas9 tools in both retinal organoids and mouse retinas. These delivery patterns and editing efficiencies remained consistent across dual adeno-associated virus (AAV) systems and transiently delivered ribonucleoprotein complexes. Our findings demonstrate that retinal organoids achieve editing outcomes comparable to those observed in vivo underscoring their utility as part of a preclinical testing platform for genome editing, with implications for advancing gene therapy research in inherited retinal diseases.