DM domain transcription factors play important roles in sexual development in a wide variety of species from invertebrate to humans. Among seven mammalian family members of DM domain transcription factors, DMRT1 has been studied in mouse and human for its conserved role in male gonadal identity. Chromosomal deletion of 9p24.3, the region in which is located, is associated with 46,XY gonadal dysgenesis. knockout (KO) mice also showed male-to-female gonadal reprogramming. However, the phenotype of KO mouse appears only after birth while 46,XY gonadal dysgenesis occurs during the developmental phase, and the cause behind this difference remained unknown. We hypothesized that in human the function of other genes clustered with , namely , might also be impaired by the chromosomal deletion, which leads to the gonadal dysgenesis phenotype. Thus, simultaneous loss of multiple DM domain genes in mice could have a more severe impact on gonadal development. To address this issue, we generated double KO mice for and via the CRISPR/Cas9 system. Comparing adult and neonatal testes of single and double KO mice, we found that loss of or , or both, does not have apparent effect on male gonadal formation during embryonic development. Our study demonstrated that the discrepancy between human with 9p24.3 deletion and KO mouse could not be explained by the simultaneous loss of gene. CRISPR/Cas9 is a versatile and straightforward approach to elucidate the questions that were otherwise difficult to address with conventional methods.