Intracellular protein editing enables incorporation of noncanonical residues in endogenous proteins

The ability to study proteins in their native cellular context is crucial to our understanding of biology. In this work, we report a technology for intracellular protein editing, drawing from split intein–mediated protein splicing, genetic code expansion, and endogenous protein tagging. This approach enables us to rapidly and site-specifically install residues and chemical handles into a protein. We demonstrate the power of this platform to edit cellular proteins, inserting epitopes, protein-specific sequences, and noncanonical amino acids. Notably, we use an endogenous tagging approach to apply our protein editing technology to endogenous proteins with minimal perturbation. We anticipate that the protein editing technology presented in this work will be applied to a diverse set of problems and phenomena in live mammalian cells.