Directed evolution of a bacterial leucyl tRNA in mammalian cells for enhanced noncanonical amino acid mutagenesis
The E. coli leucyl-tRNA synthetase (EcLeuRS)/tRNA EcLeu pair has been engineered to geneticallyencode a structurally diverse group of in yeast but he poor activity of tRNA EcLeu has been limited in mammalian cells. We evolved tRNA EcLeu directly in mammalian cells using a virus-assisted selectionscheme to overcome this limitation and resulted in the identification of several variants with remarkably improved efficiency for incorporating a wide range of ncAAs. The advantage of the evolved leucyl tRNAs was demonstrated by expressing ncAA mutants in mammalian cells that were challenging to express before using the wild-type tRNA EcLeu , by creating viral vectors that facilitated ncAA mutagenesis at a significantly lower dose, and by creating more efficient mammalian cell lines stably expressing the ncAA-incorporation machinery.