A method has been developed to expand the genetic code of living cells to incorporate non-canonical amino acids (ncAAs) into proteins, including β-amino acids, α,α-disubstituted amino acids, and β-hydroxy acids. This approach involves the creation of orthogonal aminoacyl-tRNA synthetase (aaRS)-orthogonal tRNA pairs that can specifically recognize and incorporate ncAAs into proteins. The key innovation is the use of tRNA display, which enables the direct selection of aaRSs that can acylate their cognate orthogonal tRNAs with ncAAs, regardless of whether these ncAAs are ribosomal substrates. This method overcomes the evolutionary deadlock that has limited the genetic code to α-L-amino acids and related hydroxy acids. By using tRNA display, researchers have successfully selected aaRSs that can incorporate eight different ncAAs, including β-amino acids and α,α-disubstituted amino acids, into proteins. This allows for the site-specific incorporation of these ncAAs into proteins, expanding the chemical scope of the genetic code. The study also demonstrates the ability to encode these ncAAs into proteins in living cells, with the structure of a β-amino acid-containing protein being solved. The findings highlight the potential of this method to enable the synthesis of new classes of polymers and proteins, with applications in drug development and synthetic biology. The method is scalable and can be extended to other synthetase and tRNA systems to further expand the range of ncAAs that can be incorporated into proteins.A method has been developed to expand the genetic code of living cells to incorporate non-canonical amino acids (ncAAs) into proteins, including β-amino acids, α,α-disubstituted amino acids, and β-hydroxy acids. This approach involves the creation of orthogonal aminoacyl-tRNA synthetase (aaRS)-orthogonal tRNA pairs that can specifically recognize and incorporate ncAAs into proteins. The key innovation is the use of tRNA display, which enables the direct selection of aaRSs that can acylate their cognate orthogonal tRNAs with ncAAs, regardless of whether these ncAAs are ribosomal substrates. This method overcomes the evolutionary deadlock that has limited the genetic code to α-L-amino acids and related hydroxy acids. By using tRNA display, researchers have successfully selected aaRSs that can incorporate eight different ncAAs, including β-amino acids and α,α-disubstituted amino acids, into proteins. This allows for the site-specific incorporation of these ncAAs into proteins, expanding the chemical scope of the genetic code. The study also demonstrates the ability to encode these ncAAs into proteins in living cells, with the structure of a β-amino acid-containing protein being solved. The findings highlight the potential of this method to enable the synthesis of new classes of polymers and proteins, with applications in drug development and synthetic biology. The method is scalable and can be extended to other synthetase and tRNA systems to further expand the range of ncAAs that can be incorporated into proteins.