CD8+ T cells play a critical role during adaptive immune response, which often change locations and expand or contract in numbers under different states. In the past, many attempts to develop CD8+T cells that express luciferase in vivo have involved the use of viral transduction, which has drawbacks of hardly tracked via detection of luciferase signal in untouched natural states. Here, we generate a transgenic mouse model via CRISPR-mediated genome editing, C57BL/6-CD8a em(IRES-AkaLuci−2A-EGFP) knock-in mice(CD8a-Aka mice), as a novel tool for non-invasive imaging of CD8+ T cells, which expressed a highly sensitive luciferase-Akaluciferase. Our study offers a convenient and robust tool for understanding fundamental CD8+ T cell biology in experimental applications and preclinical translational studies. • Generate C57BL/6-CD8a em(IRES-AkaLuc −2A-EGFP) knock-in mice. • Aka-luciferase expression offers superior sensitivity to track CD8+ T cells in vivo. • Higher Akaluciferase expression signals found in "hot" solid tumor model established in CD8a-Aka mice compared with "cold" ones. • Aka-luciferase expression in CD8a-Aka mice is responsive to checkpoint blockade in the murine MC38 colorectal carcinoma model. [ABSTRACT FROM AUTHOR]