Cell therapy is one of the newest therapeutic approaches for treating tissue destruction diseases and replacing damaged parts in defective tissues. Among different cells, mesenchymal stem cells (MSCs) have received a lot of attention due to their advantages and desirable properties. Also, MSCs-derived secretome, which includes various growth factors, cytokines, and extracellular vesicles (EVs), is used in the treatment of different types of diseases. However, the application of MSCs in an intact form brings their functionality with limitations. For this reason, different methods are recommended to increase their efficiency and the extracellular vesicles derived from them. One of these methods is gene editing of these cells. Among the different techniques for MSCs gene editing, CRISPR/Cas9 can increase the therapeutic potential of MSCs in a targeted manner due to its advantages. In order to achieve the desired result, various genes have been manipulated in MSCs, including genes involved in stemness, aging, migration, proliferation, survival, and inflammatory responses. Engineering MSCs with this method affects the cells' characteristics, changes their cytokine and different growth factors secretions, and increases their therapeutic efficiency.