PROJECT SUMMARY Gene-augmentation therapy cannot address autosomal-dominant (ad) disorders, in which a defective protein is expressed. To treat these mutations, such as the dominant form of rhodopsin (RHO)-related retinitis pigmentosa RP (adRP), the only cure is to silence (RNAi) or edit the mutant allele. Precise CRISPR therapeutic editing is predicted to be the best means of treating several dominant disorders, but molecular methods harnessing CRISPR have been slow to develop because homology-directed repair (HDR) occurs only during the S and G2 phases of the cell cycle. To overcome these limitations, we have devised a novel method that capitalizes on single nucleotide polymorphism (SNP) heterozygosity to create a system applicable to multiple mutations, referred to hereafter as SNP editing. To allow for immediate translation of editing strategies targeting human- specific sequences, we rely on our recently engineered, humanized RHO adRP mouse model (RHOP347L). We aim to assess and compare our proposed SNP editing with our previously established ablate-and-replace strategy capable of overcoming challenges associated with the mutation-specific targeting of traditional CRISPR- Cas9 ablation in dominantly-inherited conditions. SNP editing, on the other hand, relies on the ablation of the mutant gene in an allele-specific manner via CRISPR-Cas9 targeting of heterozygous SNPs, leaving intact the WT allele to support normal function. By testing these techniques on our novel mouse line, we will validate our cost-effective SNP editing and ablate-and-replace strategies and evaluate the in vivo toxicity of SNP editing. Our critical and innovative modification promises to significantly reduce costs by overcoming the need to engineer, validate and obtain FDA-approval for all CRISPR components for each mutation (a major limitation of mutation- specific CRISPR/Cas9. To conduct preclinical evaluation of this species-specific technology, we have replaced the naïve mouse chromosomal Rho with human RHO mutant sequences (humanized RHO). If successful, the viral vectors used in our therapeutic editing strategy could be directly reused in human trials for adRP without further modification, thus avoiding FDA hurdles and accelerating translation of this research to a clinical setting.