Xuanqi Ren, Ziyang Zhou, Lingkai Kong, Yibo Guo, Yan Liu, Jiazhen Li, Xiaosong Gu, Chunping Jiang, Junhua Wu
Dysregulated immune activity is a cornerstone of numerous diseases, where excessive suppression enables cancer progression and uncontrolled activation drives autoimmunity. Immunotherapy aims to restore this balance, however, conventional approaches are often limited by suboptimal precision, efficacy, or long-term safety. The emergence of the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) genome-editing system has opened transformative avenues for overcoming these hurdles. This review traces the development of the CRISPR-Cas9 toolkit, encompassing gene knock out (CRISPR-KO), knock in (CRISPR-KI), transcriptional activation (CRISPRa), interference (CRISPRi), base editing and prime editing-and examines their application in reprogramming immune responses. We focus on how these six strategies enhance anti-tumor immunity by engineering immune cells to bypass inhibitory checkpoints and the tumor microenvironment, and suppress autoimmunity by restoring immune tolerance through precise genomic and transcriptional interventions. Finally, we discuss translational challenges and future directions, including the adoption of next-generation editors and smart delivery systems, which are poised to maximize the therapeutic potential of CRISPR-based cellular interventions.