Fig. 1: Engineering of PE and eVLP architectures.

a, Schematic of v1 PE-eVLPs. b, Prime editing efficiencies of v1 PE-eVLPs for the HEK3 +1 T-to-A edit in HEK293T cells and Dnmt1 +2 G-to-C edit in N2A cells. Adoption of epegRNAs (v1.2) and the PEmax architecture (v1.3) improved prime editing efficiencies compared to v1.1 PE-eVLPs. c, Schematic of PE engineering to eliminate the endogenous protease cleavage site (TSTLLIENS) in MMLV RT. d, Representative improvements in PE-eVLP editing efficiencies as a result of RT ___domain engineering. PEmax–FL denotes v1 PE-eVLPs with full-length MMLV RT as PE effector ___domain; PEmax–RNaseH del denotes v1 PE-eVLPs with RNaseH ___domain-truncated RT used as the PE effector ___domain; PEmax-6aa del denotes v2.1 PE-eVLPs with six-amino-acid-deleted RTs used as the PE effector ___domain. e, Schematic of the proposed mechanism of eVLP maturation and cargo delivery, and the design of v2.2 and v2.3 PE-eVLPs. f, Prime editing efficiencies of PE-eVLPs with the 3× NES placed at various locations (NES position 1–5) of the Gag ___domain. NES position 5 (v2.2) showed improved editing compared to that of v2.1 PE-eVLPs. g, Comparison of prime editing efficiencies with v1, v2.1, v2.2 and v2.3 PE-eVLPs at the HEK3 locus in HEK293T cells and Dnmt1 locus in N2A cells. Values shown in all graphs represent the mean prime editing efficiencies ± s.e.m. of three biological replicates. Data were fitted to four-parameter logistic curves using nonlinear regression. Comparisons of different versions of PE-eVLPs are made with eVLPs produced and transduced in parallel in one large experiment to minimize variability between preparations. Data from all PE-eVLPs produced and tested in parallel are provided in Supplementary Fig 1. The graphs in b, d, f and g show a subset of data from Supplementary Fig 1. For all conditions, 30,000–35,000 cells were treated with eVLPs containing ~2.5 × 10⁸ eVLPs μl⁻¹.