A gradient phage-assisted continuous evolution method for screening suppressor tRNAs in Escherichia coli

Suppressor tRNAs, notable for their capability of reading through the stop codon while maintaining normal peptide synthesis, are promising in treating diseases caused by premature termination codons (PTC). However, the lack of effective engineering methods for suppressor tRNAs has curtailed their application potential. Here, we introduce a directed evolution technology that employs phage-assisted continuous evolution (PACE), combined with gradient biosensors featuring various PTCs in the M13 gene III. Utilizing this novel methodology, we have successfully evolved tRNA^{Trp (UGG)} reading through the UGA stop codon in Escherichia coli. Massively parallel sequencing revealed that these mutations predominantly occurred in the anticodon loop. Finally, two suppressor tRNA^{Trp (UGA)} mutants exhibited over fivefold increases in readthrough efficiency.