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

Human retinal organoids are <i>in vitro</i> 3D structures that recapitulate key molecular and structural characteristics of the <i>in vivo</i> 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 <i>RHO</i> gene implicated in autosomal dominant retinitis pigmentosa (adRP). Using retinal organoids, <i>in vitro</i> HEK293T cells, and two humanized mouse models carrying different <i>RHO</i> 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 <i>in vivo</i>. 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 <i>in vivo</i> underscoring their utility as part of a preclinical testing platform for genome editing, with implications for advancing gene therapy research in inherited retinal diseases.