There is a fundamental gap in treatment for Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). Pharmacological treatment of CPVT is partially effective for a disease that causes sudden death. A fuller understanding of novel gene editing approaches is needed to develop safe, non-surgical, and effective therapies that target the underlying causes of CPVT. In approximately 60% of CPVT patients, mutations in RYR2 cause arrhythmia through abnormal calcium handling in cardiomyocytes. The lab has shown that using CRISPR/Cas9 to knockdown the RYR2 mutant allele is effective in preventing ventricular tachycardia(VT) by decreasing expression of dysfunctional RyR2. The overall objective of this application is to improve therapy for patients with CPVT. Preliminary data generated by the applicant showed that directly targeting a disease- causing mutation with traditional CRISPR/Cas9 therapy in a CPVT mouse model prevented pacing induced VT but reduced total expression of RyR2. The central hypothesis of this proposal is that novel methods of CRISPR/Cas9 gene editing can treat CPVT by specifically correcting causative mutation sites, reducing mutant RyR2 expression while preserving total RyR2 expression, normalizing Ca2+ handling, and decreasing susceptibility to VT. The rationale for this research is that an understanding of the effectiveness and specificity of gene editing in the correcting mutations in the heart may lead to safe novel approaches to treat genetic cardiac disorders. The hypothesis will be tested with the following specific aims 1) test the therapeutic potential of human RYR2 mutation correction in an iPSC-CM preclinical model of CPVT and 2) test the therapeutic potential of mouse RYR2 mutation correction in a mouse model of CPVT. To determine aim 1, we deliver CRISPR/Cas9 prime editing vectors to IPSC, derive CM, and measure function through confocal and light sheet imaging of IPSC and 3D cardiac organoids. To determine aim 2, we will use Lenti-CRISPR/Cas9 prime editing vectors to treat mouse models of CPVT and measure long-term cardiac function through echocardiography, EKG, programmed electrical stimulation, and molecular analysis. This research is significant in that it will advance gene editing correction of RYR2 mutations as a safe and effective method for the treatment of CPVT.