Engineering immune-boosting cancer therapy via CRISPR/Cas9 gene editing and enzyme-catalyzed prodrug activation

Overcoming tumor immunosuppression remains a major obstacle that limits the efficacy of current therapies. At the same time, there is an urgent need for strategies that achieve precise and synergistic treatment with high selectivity and minimal systemic toxicity. Here, we present a multifunctional nanoplatform denoted as ZHR, which is based on 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)-doped metal–organic frame works (MOFs) and co-delivers horseradish peroxidase (HRP) and CRISPR/Cas9 ribonucleoprotein (RNP) com plexes targeting PD-L1 and CD47. Activated by elevated hydrogen peroxide (H2O2) in the tumor microenvironment (TME), HRP catalyzes indole-3-acetic acid (IAA) to produce reactive oxygen species (ROS) for direct cytotoxicity, while ABTS radicals simultaneously generate photothermal effects that induce immunogenic cell death (ICD) and promote calreticulin (CRT) exposure to enhance immune recognition. Concurrently, CRISPR/Cas9-mediated disruption of PD-L1 and CD47 abrogates immunosuppressive signaling through the PD- L1/PD-1 and CD47/signal regulatory protein α (SIRPα) pathways, restoring T-cell activity and macrophage phagocytosis. By integrating enzyme-catalyzed prodrug activation, photothermal ICD induction, and immune checkpoint editing, this platform enables precise tumor-targeted therapy while ensuring high biosafety, simul taneously achieving tumor suppression, immune activation, and checkpoint blockade reversal. This work offers a promising strategy for synergistic immunotherapy and provides valuable insights for the design of TME- responsive, multimodal therapeutic systems.