30Premature termination codons (PTCs) are responsible for 10-15% of all inherited disease. PTC 31 suppression during translation offers a promising approach to treat a variety of genetic disorders, yet 32 small molecules that promote PTC read-through have yielded mixed performance in clinical trials. 33We present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs 34 (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino 35 acid. In total, we identified ACE-tRNA with a high degree of suppression activity targeting the most 36 common human disease-causing nonsense codons. Genome-wide transcriptome ribosome 37 profiling of cells expressing ACE-tRNA at levels which repair PTC indicate that there are limited 38 interactions with translation termination codons. These ACE-tRNAs display high suppression 39 potency in mammalian cells, Xenopus oocytes and mice in vivo, producing PTC repair in multiple 40 genes, including disease causing mutations within the cystic fibrosis transmembrane conductance 41 regulator (CFTR). 42 43 44 Amino acid codons most vulnerable to PTC conversion are those with a single nucleotide 56 substitution from a stop codon: tryptophan, tyrosine, cysteine, glutamic acid, lysine, glutamine, 57 serine, leucine, arginine, and glycine (Supplemental Figure 1). As such, PTCs represent a unique 58 constellation of diseases which afflict over 30 million people worldwide, accounting for 10-15% of all 59 genetic diseases 13 . 60 61 Small molecules, such as aminoglycosides 14 , dipeptides 15 , and oxadiazoles 16 , promote the "read-62 through" or "suppression" of nonsense mutations. These compounds are effective in model 63 organisms 17, 18 , mammalian cell lines 19 and some animal disease models 16, 20 . However, this 64 approach results in the encoding of a near-cognate amino acid 21 , effectively generating a missense 65 mutation at the PTC, which itself may have deleterious effects on protein folding, trafficking, and 66 function. Furthermore, aminoglycosides are oto-and nephrotoxic 22 , and the first-in-class oxadiazole, 67Ataluren, displayed unexpectedly low efficacy in patient populations (ACT DMD Phase 3 clinical trial, 68 NCT01826487; ACT CF, NCT02139306), thus limiting their utility as PTC therapeutics. Recent and 69 ongoing advances in CRISPR/Cas9-mediated genome editing provides potentially a permanent 70 solution for diseases resulting from nonsense mutations. However, aspects of this technology 71 impart hurdles for its rapid use as a therapeutic 23, 24 , and these challenges are not limited to the 72 requirement of "precision" or "personalized" diagnostics for each mutation based on the context of 73 each patient's genetic variability. 74
75We sought to identify a PTC repair approach that displays the versatility of small molecules and the 76 precision of gene editing. We investigated tRNAs to fulfill these criteria, whereby their anticodons 77 have been engineered via mutagenesis to recognize and suppress UGA, UAA or U...