equip eukaryotic cells with dual orthogonal expanded genetic codes that enable the specific reprogramming of distinct translational machineries with single-residue precision. [14] Tharp et al. demonstrated that UAU can be reassigned along with UAG or UAA to encode two distinct ncAAs in the same protein. [15] In addition, the same ncAA has been inserted into the same protein at several TAG sites by deleting RF1 in E. coli or engineering eukaryotic release factor 1 (eRF1) in mammalian cells. [16][17][18][19] However, the site-specific incorporation of three distinct ncAAs into a single mammalian protein remains a challenge.Effective and high-fidelity incorporation of three distinct ncAAs into a single mammalian protein will greatly enhance the investigation of protein structures and functions, [10,20,21] including the site-specific mimicry of posttranslational modifications and in situ labeling for multicolor imaging, among other applications. [22][23][24][25] To achieve this incorporation, ncAAs must be delivered by bio-orthogonal aaRS/ tRNA pairs that do not cross-react with each other or their counterparts in host cells. [9,26] The suppression systems driving the incorporation of three distinct ncAAs can cross-react at three different levels. First, an aaRS might charge the substrate ncAA for another aaRS. Second, an aaRS might charge a noncognate tRNA. Finally, a suppressor tRNA might recognize a noncognate nonsense codon. Four distinct aaRS/tRNA pairs have previously been used to expand the genetic code of mammalian cells including a tyrosyl (EcTyr) and leucyl (EcLeu) pair derived from E. coli in addition to an M. mazei pyrrolysyl (MmPyl) pair and an M. barkeri pyrrolysyl (MbPyl) pair. [10,21,22] In addition, the PylRS from Methanomethylophilus alvus has been used extensively in recent years to install UAAs into proteins in eukaryotes. [12] However, the MmPyl and MbPyl pairs cannot be used together because they are both derived from Methanosarcina and share similar synthetase structures. Thus, it is possible, in principle, to combine the EcTyr and EcLeu pairs with either the MmPyl or MbPyl pairs to incorporate three distinct ncAAs into a single mammalian protein.In this study, a systematic approach was used to select the optimal combination of three orthogonal ncAA incorporation systems based on read-through efficiency and orthogonality. Specifically, EcLeuRS/tRNA CUA , MmPylRS/tRNA UCA , and EcTyrRS/tRNA UUA were used to simultaneously incorporate three distinct ncAAs into mammalian proteins by suppressing Site-specific incorporation of distinct noncanonical amino acids (ncAAs) into proteins via genetic code expansion in mammalian cells represents a new avenue for protein engineering. Reassigning three TAGs with the same ncAA in mammalian cells has previously been achieved using translational machinery. However, simultaneous recoding of three nonsense codons with distinct ncAAs in mammalian cells remains a challenge due to low incorporation efficiencies. Here, three optimized aaRS/tRNA pairs (i.e., the E. col...