Fig. 2: Engineering of pegRNA and ngRNA.

a, A dual transfection/transduction experiment identifies epegRNAs as the limiting component in the v2.3 PE-eVLPs. b, Schematic of v3 PE-eVLPs utilizing the MCP–MS2 strategy for the recruitment of epegRNAs. The incorporation of MCP–MS2 strategy enables three modes of guide RNA loading into eVLPs: (i) via binding to PE, (ii) via MS2 stem–loop binding to MCP and (iii) via MCP:MS2-gRNAs:PE three-component interaction. c, Editing efficiencies of v2.3 PE-eVLPs at the Dnmt1 locus in N2A cells with MS2 stem–loop insertion at various locations in epegRNAs. The 3′ end denotes v2.3 PE-eVLPs with insertion of the MS2 stem–loop after the structured tevoPreQ1 motif of the epegRNA; 3′ end* denotes v2.3 PE-eVLPs with insertion of the MS2 stem–loop directly after the 3′ extension of the pegRNA, thereby using the MS2 stem–loop to mimic a structured motif at the 3′ end of epegRNAs; TL denotes v2.3 PE-eVLPs with insertion of the MS2 stem–loop within the tetraloop of the pegRNA scaffold; and ST2 denotes v2.3 PE-eVLPs with insertion of the MS2 stem–loop within the ST2 loop of the pegRNA scaffold. d, Heatmap of editing efficiencies from stoichiometry optimization of Gag–Pol, Gag–MCP–Pol and Gag–PE plasmids for production of v3 PE-eVLPs. e, Comparison of editing efficiencies with v2.3 and v3 PE-eVLPs at the Dnmt1 locus in N2A cells. f, Editing efficiencies of all-in-one and separate-particle v3 PE3-eVLP systems with varying MS2-epegRNA to MS2-ngRNA ratios. g, Quantification of the number of epegRNAs molecules packaged per eVLP in successive generations of PE-eVLPs. h, Quantification of the number of PE protein molecules packaged per eVLP in successive generations of PE-eVLPs. Values represent the mean prime editing efficiencies ± s.e.m. of three biological replicates (a and c–f) or three technical replicates (g and h). Data were fitted to four-parameter logistic curves using nonlinear regression. Data from all PE-eVLPs produced and tested in parallel are provided in Supplementary Fig 2. The graphs in c, d and e show a subset of data from Supplementary Fig 2. For all conditions, 30,000–35,000 cells were treated with eVLPs containing ~2.5 × 10⁸ eVLPs μl⁻¹.