Intracellular Delivery of CRISPR/Cas9 via Living Bacteria to Block Arginine Metabolic Reprogramming and Enhance Tumor Chemotherapy Sensitivity

Abstract Chemotherapy, the primary treatment for triple‐negative breast cancer (TNBC), is frequently compromised by reduced sensitivity associated with amino acid metabolic reprogramming. Here, it is found that chemotherapy significantly upregulates the expression of arginine transporter SLC7A2, supporting tumor cell survival in TNBC mice and patients. The Slc7a2 gene is thus purposefully knocked down via the CRISPR/Cas9 system to enhance chemotherapy efficacy by regulating arginine metabolism. A self‐guiding living bacteria system is engineered for precise CRISPR/Cas9 transport, capable of tumor intracellular colonization and achieving endo‐/lysosomal escape. The engineered bacteria (PDC@V) selectively targeted hypoxic tumor regions in vivo, and exhibited an intracellular invasion efficiency 23.7‐fold higher than that of nonintracellular bacteria. Their inherent ability to escape the endo‐/lysosome ensured the CRISPR/Cas9 plasmids are efficiently released in response to cytoplasmic esterase activity, leading to a five fold increase in gene editing efficiency. This self‐guiding living system restricted tumor arginine uptake, alleviating resistance to the chemotherapeutic agent DOX and achieving a tumor inhibition rate 3.2 times greater than the DOX alone group. Furthermore, this strategy activated both the innate and adaptive immune systems. Together, the study presents a novel approach for delivering gene‐editing tools and highlights the potential of targeting arginine metabolic reprogramming to sensitize tumor chemotherapy.