Genetic code expansion via stop codon suppression is a powerful strategy to engineer proteins. Suppressor tRNAs are aminoacylated with noncanonical amino acids (ncAAs) by dedicated aminoacyl-tRNA synthetases (aaRS) and direct ncAA incorporation site-specifically during translation. These pairs of tRNA/aaRS must be orthogonal to the host's tRNAs, aaRS and natural amino acids. Pyrrolysyl-tRNA (PylT)/PylRS pairs from methanogenic archaea, as well as engineered tRNA/aaRS pairs derived from bacteria, are used for genetic code expansion in mammalian cells. Amber suppression is routinely achieved by transient introduction of the components leading to short-term and heterogeneous expression. Here, we demonstrate that stable integration of tRNA/aaRS genes allows for efficient, genetically encoded ncAA incorporation in diverse mammalian cell lines. We extend a general plasmid design and PiggyBac (PB) integration strategy developed for the Methanosarcina mazei PylT/PylRS pair to genomic integration of two tRNA/aaRS pairs of bacterial origin. We further explore suppression of ochre and opal stop codons and parallel incorporation of two distinct ncAAs, both accessible for click chemistry, by dual suppression in stable cell lines. Clonal selection allows for isolation of cells with high dual suppression efficiency and dual site-specific fluorescent labeling of a cell surface receptor using bioorthogonal click chemistries on live mammalian cells.

No abstract