This study presents a method for engineering carboxysome shells with programmable cargo targeting for use in protein nanocages. The researchers utilized an α-carboxysome shell in conjunction with SpyTag/SpyCatcher and coiled-coil protein coupling systems to generate a range of synthetically engineered nanocages with site-directed cargo loading. The study demonstrates that the cargo-docking sites and capacities of the carboxysome shell-based protein nanocages can be precisely modulated by selecting specific anchoring systems and shell protein domains. The findings provide insights into the encapsulation principles of the α-carboxysome and establish a foundation for the bioengineering and manipulation of nanostructures capable of capturing cargos and molecules with exceptional efficiency and programmability. The study also highlights the potential of these nanocages in applications such as catalysis, delivery, and medicine. The researchers developed 16 different types of recombinant α-carboxysome shells, in which the exogenous ST/SC and coiled-coil systems were fused at four distinct insertion sites of WT CsoS1A and circularly permuted CsoS1A^P. The results show that 11 types of ST/SC and CC^A/CC^B fusions on CsoS1A or CsoS1A^P could lead to the formation of stable shell structures with a diameter of 90–120 nm. The study also reveals that these custom-engineered shells exhibited improved capacities of recruiting GFP as non-native cargos into or onto the shell structures. The findings suggest that the ST/SC system has advantages over the coiled-coil system, and the diverse cargo-loading capacities indicate the versatility and fine-tunability of cargo loading and capture of these generated nanocages, which have significant potential in biotechnological and biomedical applications.This study presents a method for engineering carboxysome shells with programmable cargo targeting for use in protein nanocages. The researchers utilized an α-carboxysome shell in conjunction with SpyTag/SpyCatcher and coiled-coil protein coupling systems to generate a range of synthetically engineered nanocages with site-directed cargo loading. The study demonstrates that the cargo-docking sites and capacities of the carboxysome shell-based protein nanocages can be precisely modulated by selecting specific anchoring systems and shell protein domains. The findings provide insights into the encapsulation principles of the α-carboxysome and establish a foundation for the bioengineering and manipulation of nanostructures capable of capturing cargos and molecules with exceptional efficiency and programmability. The study also highlights the potential of these nanocages in applications such as catalysis, delivery, and medicine. The researchers developed 16 different types of recombinant α-carboxysome shells, in which the exogenous ST/SC and coiled-coil systems were fused at four distinct insertion sites of WT CsoS1A and circularly permuted CsoS1A^P. The results show that 11 types of ST/SC and CC^A/CC^B fusions on CsoS1A or CsoS1A^P could lead to the formation of stable shell structures with a diameter of 90–120 nm. The study also reveals that these custom-engineered shells exhibited improved capacities of recruiting GFP as non-native cargos into or onto the shell structures. The findings suggest that the ST/SC system has advantages over the coiled-coil system, and the diverse cargo-loading capacities indicate the versatility and fine-tunability of cargo loading and capture of these generated nanocages, which have significant potential in biotechnological and biomedical applications.