Tumors and cancers are found across all vertebrates. Likewise, the detection and destruction of cancerous cells is a function conserved in non-mammalian vertebrates. In human and murine cancer, the relevance of natural killer T (NKT) in tumor immunity is an active research area. Notably, the conditions that lead to either anti- or pro-tumoral activities of these cells remain partially understood. To help resolve this issue, we are taking a comparative and evolutionary approach that relies on a tumor immunity model in the amphibian Xenopus. Despite phylogenetic distance, the immune system between amphibians and mammals is fundamentally similar. Using RNA interference (RNAi), we demonstrated that one of Xenopus MHC class I-like (XNC) genes, XNC10, is critical for the development and tumor-related function of Vα6 innate-like T (iT) cell subset, expressing an invariant T cell receptor (iTCR) α chain rearrangement – Vα6-Jα1.43. Our recent study suggests that an additional iT cell subset (Vα22 iT), characterized by Vα22-Jα1.32 iTCR α chain, is specifically involved in the immunity against Xenopus thymic lymphoid tumors. The intraperitoneal transplantation of Xenopus tumor cells into syngeneic tadpoles results in significant early infiltration of both Vα6 and Vα22 iT cells, concomitant with the decrease of these subsets from spleen and thymus. We hypothesize that the recruitment and anti-tumor activity of the two iT cell subsets can be manipulated by impairing the tumor expression of XNC10 and/or other XNC genes. To explore this possibility, we are developing a reverse genetic approach with RNAi and three-component CRISPR/Cas9 system to generate transgenic animals deficient in either Vα6 or Vα22 iT cells and tumor lines stably deficient in specific XNC gene.