SUMMARY Inherited retinal disorders are a genetically heterogeneous group of blinding diseases that have significant impact on quality of life. Therapeutic approaches have lagged significantly behind initial identification of the genetic bases for these diseases. However, there are some striking successes; e.g., RPE65 gene augmentation therapy was the first FDA-approved gene therapy for any genetically inherited disease. Clinical translation of current CRISPR-Cas9 technology has been impeded by its low editing efficiency, error-prone homology-directed repair (HDR), and substantial indel formation. Precision genome editing is an advanced, innovative CRISPR-Cas9- associated genome-editing tool that addresses the limitations of typical CRISPR-Cas9 implementation. Adenine base editors (ABEs) enable conversion of a point mutation independently of Cas9-induced double-stranded DNA breaks and HDR. When base editing is not applicable (e.g., due to transversion mutations, large deletions, or insertions), prime editing technology offers feasible alternatives. Genome editing is highly specific; however, prolonged expression of base editors could lead to undesired off-target alterations throughout the genome and transcriptome. We hypothesize that transient delivery of genome editors via RNPs and synthetic RNAs can achieve the same high editing rates as those for genome editors delivered via viral transduction with reduced off-target and bystander editing. Accordingly, we propose two thematically linked aims. Aim 1. Correct inherited retinal disease-causing mutations in the rhodopsin gene (RhoE150K/E150K) associated with autosomal recessive retinitis pigmentosa (RP) via adenine base editing. Delivery of ABEs will be optimized in the thoroughly characterized RhoE150K/E150K mouse model of RP. Proposed approaches will provide a platform for ABEs to be quickly adapted to any suitable RPE or retinal mutation. Aim 2. Repair the ABCA4 protein in Abca4PV/PV mice by prime editing. Using the PE3b prime editor and two concurrent stabilized engineered prime-editing guide RNAs (epegRNA), we will restore functional ABCA4 protein in Abca4PV/PV mice that carry double allelic mutations in photoreceptors and the RPE. Using immunoblotting and next-generation sequencing for detecting rescued Abca4, and two-photon imaging techniques to detect A2E, we will optimize genome editing efficiency in this animal model to improve prime-editing technology and its application to treat inherited retinal diseases. For both aims, we will test various means to deliver the editors transiently: (i) cell-penetrating peptides fused to editors in purified ribonucleoprotein (RNP)-editing complexes; (ii) Coomassie-lipid tags on purified RNP-editing complexes; (iii) viral-like particles containing RNP-editing complexes; or (iv) lipid nanoparticles containing stabilized mRNAs of genome-editing materials for intracellular expression. These delivery systems will be optimized first in engineered chromogenic...