Intracellular organelles are subsystems within a cell, whose activity and chemical composition reflect the metabolic state of live cells. Alterations in cellular homeostasis occurring in disease, ageing and development are also reflected at the level of organelles. By targeting organelles with pharmacological agents and genetic tools, the aim is to improve disease diagnosis and to restore cell function. In this Review, we discuss biological pathways that can be exploited to target the delivery of exogenous cargo with organelle-level precision. We investigate how these pathways can be leveraged for imaging, diagnosis and therapy at the organelle level, and highlight the potential of nucleic acids as delivery systems to target specific organelles in vivo, including the nucleus, lysosomes, secretory organelles and mitochondria. The programmability, modularity and biocompatibility of nucleic acid-based scaffolds make them well suited to accomplishing next-generation targeting with organelle-level resolution in living organisms.Organelles are compartments inside cells that play important parts in intercellular and intracellular communication, reflecting the metabolic state of the cell. This Review discusses biological pathways that can be exploited to target cargo to specific organelles in vivo, highlighting nucleic acids as suitable delivery vehicles for organelle-level imaging, diagnosis and therapy.