Strategies to Expand the Genetic Code of Mammalian Cells

Genetic code expansion (GCE) in mammalian cells has emerged as a powerful technology for investigating and engineering protein function. This method allows for the precise incorporation of a rapidly growing toolbox of noncanonical amino acids (ncAAs) into predefined sites of target proteins expressed in living cells. Due to the minimal size of these genetically encoded ncAAs, the wide range of functionalities they provide, and the ability to introduce them freely at virtually any site of any protein by simple mutagenesis, this technology holds immense potential for probing the complex biology of mammalian cells and engineering next-generation biotherapeutics. In this review, we provide an overview of the underlying machinery that enables ncAA mutagenesis in mammalian cells and how these are developed. We have also compiled an updated list of ncAAs that have been successfully incorporated into proteins in mammalian cells. Finally, we provide our perspectives on the current challenges that need to be addressed to fully harness the potential of this technology.