In the new area of genetic screening, the number of variation/mutations identified in genes of unknown function in patients affected by neuromuscular diseases is growing. There is therefore a rising need for the development of tools to understand the function of those genes and to shed light on the pathophysiological mechanisms . Here we described the development, characterization and functional validation of skeletal muscle cell lines deleted of the ryanodine receptor RYR1 gene, created using lentiviruses encoding the CRISPR/Cas9 tools.