Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Genetic code expansion via stop codon suppression is a powerful strategy to engineer proteins. Pyrrolysyine-tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) pairs from methanogenic archaea and engineered bacterial tRNA/aminoacyl-tRNA synthetases (aaRS) pairs are used for site-specific incorporation of noncanonical amino acids (ncAAs) in response to stop codons in mammalian cells. Routinely, ncAA incorporation is achieved by transient expression of the tRNA/aaRS pair leading to heterogeneous suppression. Genomic integration of tRNA/aaRS expression cassettes for more homogenous, adjustable and reproducible levels of protein, containing one or more ncAA, will greatly benefit protein engineering, chemical control and imaging applications in mammalian cells. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal tRNAPyl/PylRS or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient, genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore simultaneous suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual fluorophore labeling of a cell surface receptor on live mammalian cells.