Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9), as a powerful genome-editing tool, has revolutionized genetic engineering. It is widely used to investigate the molecular basis of different cancer types. In this review, we present an overview of recent studies in which CRISPR/Cas9 has been used for the identification of potential molecular targets. Based on the collected data, we suggest here that CRISPR/Cas9 is an effective system to distinguish between mutant and wild-type alleles in cancer. We show that several new potential therapeutic targets, such as CD38, CXCR2, MASTL, and RBX2, as well as several noncoding (nc)RNAs have been identified using CRISPR/Cas9 technology. We also discuss the obstacles and challenges that we face for using CRISPR/Cas9 as a therapeutic.