Abstract Inherited retinal degenerations are a genetically heterogeneous group of disorders characterized by death of the light-sensing photoreceptor cells of the outer neural retina. For decades, physicians and scientists have dreamed of preventing vision loss in patients affected with an inherited retinal disease via some form of gene replacement therapy. Thus, it is not surprising that the recent clinical success of AAV-mediated gene augmentation for the treatment of early stage RPE65-associated disease has been tremendously exciting, providing a new level of confidence that this approach will also be useful for the treatment of other recessive and X-linked disorders. However, AAV-mediated gene augmentation is not likely to be effective as a stand- alone treatment for the large population of patients with dominantly inherited diseases that result from mutation dependent formation of a toxic protein product (i.e. gain of function variants). To treat dominant gain of function diseases, correction of the mutant allele or suppression of the abnormal transcript will be required. Recent advances in the field of CRISPR-based genome editing have shown great promise for such applications. For instance, in a recent study, we demonstrated how the subretinal injection of a CRISPR/Cas9 construct could be used to disrupt the disease-causing Pro23His rhodopsin allele at the DNA level in pig retina via nonhomologous end joining (NEHJ). Unfortunately, the efficiency of disease allele deletion was significantly lower than that required to have a clinically significant effect. In response to this poor efficiency (and the fact that off-target effects are a real concern with in vivo genome editing), we have decided to focus this renewal application on very recently developed CRISPR-based approaches to suppress transcription and cleave specific mRNAs rather than permanent DNA modification. These newly developed approaches are significantly less likely to induce deleterious off-target genomic modifications than traditional CRISPR-based strategies. Likewise, as these approaches do not require DNA cleavage and repair they have the potential to be significantly more efficient, in turn making them more suitable for in vivo applications. The CRISPR-based approaches outlined in this application will be tested in patient-specific iPSC-derived retinal organoids in vitro and the Pro23His rhodopsin mutant pig in vivo. The studies in this proposal will evaluate the feasibility of using this new dimension of CRISPR biology for the treatment of autosomal dominant retinal diseases.