Figure 2.

Generation of a minimal PE by truncating M-MLV RT

(A) Schematic of PE^CO and truncated PE^CO variants used in this study. The RT is composed of a polymerase and an RNase H domain. Initially, a truncated PE was made with the RNase H domain deleted (471 bp) to form PE^CO-ΔR. Subsequently, serial 30-bp truncations at both ends were tested with the aim of generating a minimal PE^CO (PE^CO-Mini). (B) Minimizing M-MLV RT by deleting the RNase H domain (PE^CO-ΔR) and by further N- and C-terminal trimming is shown. The same molar amounts of PE^CO-ΔR and trimmed PE^CO-ΔR plasmids were transfected into HEK293T cells together with fixed amounts of plasmids encoding pegRNA and ngRNA. Cells were subjected to ICE analysis for evaluating prime editing activity 3 days post-transfection. (C) Combinatorial N- and C-terminal truncations were analyzed for prime editing efficiencies as in (B). Based on the activity, we selected RT-ΔR with ΔN60 + C90 as the minimal RT, which is 1,410 bp long. (D) Comparison of PE^CO and the two truncated variants PE^CO-ΔR and PE^CO-Mini performed as in (B) is shown. Bars represent mean values ± SD with all data points from independent experiments shown. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.