Nature regulates interference between cellular processes-allowing more complexity of life-by confining specific functions to organelles. Inspired by this concept, we designed an artificial organelle dedicated to protein engineering. We generated a membraneless organelle to translate only one type of messenger RNA-by recruiting an RNA-targeting system, stop codon-suppression machinery, and ribosomes-by means of phase separation and spatial targeting. This enables site-specific protein engineering with a tailored noncanonical function in response to one specific codon in the entire genome only in the protein of choice. Our results demonstrate a simple yet effective approach to the generation of artificial organelles that provides a route toward customized orthogonal translation and protein engineering in semisynthetic eukaryotic cells.
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