Junlin Lu, Jiajian Lai, Liang Cheng, Hengji Zhan, Kaiwen Jie, Chaoying Liu, Lin Huang, Meifeng Cen, Sen Liu, Zehua Chen, Qiang Zhang, Jianhui Zhang, Jiahui Wu, Bolin Pan, Siting Chen, Jianmei Zhong, Baoqing He, Hui Li, Xu Chen, Tianxin Lin
CRISPR‒Cas systems represent powerful tools for genome regulation. However, the large size of Cas proteins limits their efficient delivery via an adeno-associated virus (AAV), thereby restricting their clinical translation. Here, we engineer the IS200/IS605 transposon-encoded nuclease TnpB, along with its ωRNA scaffold, to create an enhanced TnpB system, which serves as a compact toolkit for gene activation, genome editing, and base editing. The gene activator enTnpBa increases expression by 2889-fold with a minimized 93 nt ωRNA and robustly activates endogenous genes in mammalian cells. We develop a single-AAV-based regimen for immune activation (AAV-ImmunAct) that delivers enTnpBa to activate CXCL9, IL-15, and IFN-γ. AAV-ImmunAct effectively enhances T cell migration and activation, increases killing of cancer cell lines and patient-derived organoids, and synergizes with anti-PD-1 therapy in humanized mice. Here, we establish enTnpB as a compact and versatile platform for genome regulation and a promising tool for cancer immunotherapy. CRISPR–Cas tools enable genome regulation but are often too large for efficient AAV delivery. Here, authors engineer a compact enhanced TnpB–ωRNA system (enTnpB) as a versatile genome regulation platform and develop a single-AAV regimen, ImmunAct, to activate endogenous cytokines and enhance cancer immunotherapy.