Abstract: Objective The NOD/SCID/IL2Rγ-/- (NSG) mouse strain is the most widely used immunodeficient strain for xenograft transplantation. However, the existing SCID mutation is a spontaneous mutation of the Prkdc gene, which leads to leaky T cell developmental block and difficulty in genotyping. It is therefore important to develop a new strain of NSG mice with targeted disruption of Prkdc and IL2Rγ genes. Methods Targeted disruption of Prkdc and IL2Rγ genes was achieved using the CRISPR/ Cas9 system. By intercrossing the knockout and NOD mice, we obtained a novel strain of NOD/SCID/IL2Rγ-/- (NSG) mice, denoted as cNSG (Chinese NSG) mice. Results In addition to the NOD mutation, cNSG mice exhibited a complete absence of T cells, B cells and NK cells. cNSG mice allowed more efficient engraftment of human cancer cells than the commonly used immunodeficient nude mice. Conclusion cNSG mice will provide an important xenotransplantation model for biomedical research.