Precise RNA Editing: Cascade Self‐Uncloaking Dual‐Prodrug Nanoassemblies Based on CRISPR/Cas13a for Pleiotropic Immunotherapy of PD‐L1‐Resistant Colorectal Cancer

Abstract CRISPR/Cas13a is a powerful genome editing system for RNA knockdown that holds enormous potential for cancer treatment by targeting currently undruggable oncogenes or immune checkpoints. However, the precise intratumoral activation of CRISPR/Cas13a to maximize the therapeutic efficiency while guaranteeing biosafety remains a daunting challenge. Here, a cascade self‐uncloaking nanoassembly (SRC) based on a dual‐prodrug comprising SN38 and Cas13a/RNP is developed, and the external encapsulation is performed by coating with a ROS‐responsive probe, which is stimulated by the tumor microenvironment to achieve the efficient NIR‐II imaging by CH10055 due to disaggregation into single molecules, while the second release of prodrug in the hypoxic environment enables targeted controlled release. SN38 not only induces immunogenic cell death (ICD), but significantly combats the immunosuppressive microenvironment of colorectal cancer in combination with the RNA editing targeting the novel immune checkpoint TIM3 to regulate the cGAS‐STING pathways, resulting in synergistic activation of both innate and adaptive immunity. The treatment of SRC exhibits a tenfold increase in tumor regression of α ‐PD‐L1 in PD‐L1‐resistant orthotopic and xenograft models by inducing effective tumor immune infiltration. These results demonstrate the feasibility of using CRISPR/Cas13a in cancer treatment, and SRC holds immense promise as a neoadjuvant strategy for enhancing CRC immunotherapy.