The study explores the engineering of carboxysome shells to create protein nanocages with programmable cargo targeting. Carboxysomes, natural proteinaceous organelles found in cyanobacteria and proteobacteria, have shown potential in creating versatile nanocages due to their self-assembly, cargo encapsulation, permeability, and modularity. The researchers developed a systematic approach to generate a range of synthetically engineered nanocages with site-directed cargo loading based on an α-carboxysome shell using SpyTag/SpyCatcher and Coiled-coil protein coupling systems. They demonstrated that the cargo-docking sites and capacities of the carboxysome shell-based protein nanocages could be precisely modulated by selecting specific anchoring systems and shell protein domains. The study provides insights into the encapsulation principles of α-carboxysomes and establishes a foundation for the bioengineering and manipulation of nanostructures capable of capturing cargos and molecules with exceptional efficiency and programmability, enabling applications in catalysis, delivery, and medicine.The study explores the engineering of carboxysome shells to create protein nanocages with programmable cargo targeting. Carboxysomes, natural proteinaceous organelles found in cyanobacteria and proteobacteria, have shown potential in creating versatile nanocages due to their self-assembly, cargo encapsulation, permeability, and modularity. The researchers developed a systematic approach to generate a range of synthetically engineered nanocages with site-directed cargo loading based on an α-carboxysome shell using SpyTag/SpyCatcher and Coiled-coil protein coupling systems. They demonstrated that the cargo-docking sites and capacities of the carboxysome shell-based protein nanocages could be precisely modulated by selecting specific anchoring systems and shell protein domains. The study provides insights into the encapsulation principles of α-carboxysomes and establishes a foundation for the bioengineering and manipulation of nanostructures capable of capturing cargos and molecules with exceptional efficiency and programmability, enabling applications in catalysis, delivery, and medicine.