Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases (IRD). Gene-based therapies, mostly in the form of gene addition or supplementation therapies using adeno-associated viral (AAV) vectors for gene delivery, have been developed for several IRDs. However, all so far targeted diseases are associated with mutations in genes small enough that the corresponding cDNA can be transferred in a single AAV. Unfortunately, many of the most frequently mutated genes such as ABCA4 exceed the AAV cargo size. ABCA4 mutations cause Stargardt disease, an early onset form of macular degeneration. As gene therapies are hampered for Stargardt disease due to ABCA4 sequence length, gene editing represents an attractive approach to correct the mutation in the genomes of the patient’s photoreceptors. Precise gene editing, to avoid unwanted and uncontrolled additional genomic alterations, requires homology-dependent double strand break (DSB) repair. As DSB pathways differ according to the cell cycle’s stage, in the first SPP2127 funding period, we have demonstrated that precise DSB repair also occurs in postmitotic neurons. In addition, DSB pathway modifications further improved precise repair. We have also identified human stem cell-derived neurons as an adequate in vitro testbed for testing all experimental parameters for precise genome editing. In addition, the DSB activity in healthy and diseased human and mouse photoreceptors is at work, unaltered and shows a high activity homology to human neurons. Our data suggest that mouse models represent sophisticated in vivo models for therapeutic gene editing. Based on our findings, we will assemble all molecular tools for DSB pathway engineering, gRNAs, ABCA4 templates and DSB reporter constructs, which will be systematically applied to human induced neurons to reveal the optimal parameters for precise repair. We will also generate an ABCA4 mutated human stem cell line that we will use for generating retinal organoids. These 3D human retinal organoids contain lots of photoreceptors that we will target by AAVs to deliver all necessary components for gene correction. We will combine imaging, transcriptomic, genomic and quantitative proteomic readouts to study the ABCA4 repair in depth. Ultimately, we will test our approach also in a Stargardt disease mouse model to correct the Abca4 gene in vivo. Treated mice will be studied using live imaging, behavior testing and electrophysiology. On- and potential off-target effects will be revealed by next generation sequencing. Demonstrating in vivo efficacy and safety as well as employing sophisticated human in vitro models for correcting the ABCA4 locus streamlines and paves the way for clinical translation. Our proof-of-concept study for precise gene editing including DSB pathway engineering will also be instructive for other therapeutic interventions for IRDs but also in general for genomic engineering of postmitotic neurons.