Fig. 2: Identifying prime editing-accessible sup-tRNA mutation combinations.

a, Results of saturation mutagenesis of tRNA-Leu-TAA-4-1, with mutations that are comparable or better than an ac-only sup-tRNA shown in purple. Bases that would easily be accessible with prime editing (PE-accessible bases) at the same time as changing the anticodon are circled with a dashed line. In this representation, each hairpin dinucleotide is represented by a number (hp1–hp25). b, Heat map showing the relative fold change in activity of hairpin mutations compared with the ac-only sup-tRNA. Hairpin positions correspond with hp1–hp25 in a. c, Sequences of the anticodon stem loops of five prime editing-accessible mutation variants and an ac-only control, from which 19 variant combinations were generated. d, Protein yield relative to wild type (WT) after readthrough with an endogenously converted tRNA-Leu-TAA-1-1 sup-tRNA, a single-copy lentiviral tRNA-Leu-TAA-1-1 sup-tRNA with the indicated mutations, or with alternative engineered sup-tRNAs (described in Porter et al.³² and Wang et al.⁵). The Leu-ss94-tStem9 variant is the engineered mature tRNA sequence converted to a TAG suppressor, flanked by the immediate 55-bp leader sequence of the tRNA-Cys-GCA-12-1 gene and the 35-bp trailer sequence of the tRNA-Ile-TAT-1-1 gene. The number of bases from the nearest endogenous tRNA are indicated. Data are mean ± s.d. of n = 2 independent biological replicates.