Directed Evolution of a Bacterial Leucyl tRNA in Mammalian Cells for Enhanced Noncanonical Amino Acid Mutagenesis.

The leucyl-tRNA synthetase (EcLeuRS)/tRNA pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNA. Here, we overcome this limitation by evolving tRNA directly in mammalian cells by using a virus-assisted selection scheme.