Introduction: Advances in next generation sequencing technology has created a new clinical challenge by identifying increasing numbers of variants of unknown significance (VUS). A VUS is a rare change in the DNA sequence that, based on our current understanding, cannot be classified as pathogenic or benign, and whose contribution to disease is unknown. The VUS challenge is prominent in the case of the cardiac ryanodine receptor gene (RyR2), a large gene that encodes a protein that forms a tetrameric complex on the sarcoplasmic reticulum and has a critical role in the sarcoplasmic release of stored calcium ions in cardiac muscle. Mutations in RyR2 can cause life-threatening arrhythmias which can lead to sudden cardiac death in young and apparently healthy individuals. We have identified a VUS, R4790*, and established patient-derived induced pluripotent stem cells, from which we can derive cardiomyocytes (iPSC-CMs). Combining this iPSC technology and cutting edge CRISPR gene editing technology, to generate isogenic cell controls, could be used as a novel approach to determine VUS pathogenicity. Objective: To develop CRISPR-based strategies to either correct a RyR2 VUS or suppress the expression of the VUS allele in iPSC-CMs. iPSC-CMs were generated from a patient who suffered a cardiac arrest and carries a novel nonsense variant in the RyR2 gene (R4790*). Methods and Results: We established optimal conditions to deliver CRISPR-Cas9 gene editing components to the iPSCs through a nucleofection protocol that did not alter the pluripotent state of the cells. We also established differentiation protocols to direct the iPSC cultures into CM cultures, where calcium release could be monitored. We used several gene editing strategies to target the RyR2 gene itself in the patient derived cells, initially attempting to direct correct/mutate the residue via homology directed repair. Despite several experimental strategies (including changes in DNA repair template, use of different CRISPR-Cas enzymes: Cas9 and Cas12a), we were unable to generate sufficient CRISPR cutting efficiency at this specific region. Next, we reasoned that knock out (KO) or deletion of a single allele would allow us to investigate either the mutant or wild-type version of the protein in isolation. Thus we targeted the VUS allele by using either allele-specific or unbiased guide RNA (gRNAs). Allele specific gRNAs, target sites formed by other variants in the intronic regions of the gene, also failed to be effective. Two unbiased targeting strategies, one exploiting Non-Homologous End Joining (NHEJ) and the other Homology Directed Repair (HDR) were both successful at generating a series of clonal iPSCs of different genotypes, including full KO and heterozygote KO. The NHEJ method creates a premature STOP codon via random insertion/deletion (InDel) of nucleotides at the site of cutting, and this was confirmed by next generation sequencing in five independent clones. In these cells we identified no change in mRNA levels, but alterations in protein levels assayed by western blot. These conflicting data possibly indicate a cell adaptation to the InDel, or perhaps secondary start sites in the gene. Thus, the HDR method was designed to specifically delete ~450 kb of genomic sequence, comprising the majority of the gene sequence, whilst inserting a Puromycin selection marker to enable easy isolation of correctly targeted clones. This strategy led to RyR2 deletion of both genomic and coding DNA and a reduction in RyR2 protein (six independent clones analysed). Conclusions: This study demonstrates that CRISPR technology can edit the genome of patient derived iPSCs and can target the RyR2 gene. Several CRISPR-based approaches in generating RyR2 allele deletion were compared in the context of a human genetic background, and have led to the establishment of a series of clonal cell lines that will allow functional characterisation of the R4790* variant. This strategy could serve as a blueprint for screening and testing of other VUS in RyR2 and other genes.