Fig. 2: Prime editing (PE) vs L-PGI for installation of point mutations in 3 disease-relevant loci in wild-type primary human hepatocytes (PHH).

Results shown for n = 3 biological replicates for all experiments unless otherwise indicated. A Efficiency and fidelity of 2 nt substitution of +5 G to T and +8 G to T to install H1069Q with silent mutation in ATP7B assayed by next generation sequencing (NGS). L-PGI was compared to PE2, PE3, and PEMax. Ordinary one-way ANOVA without matching with Gaussian distribution used to calculate p value for efficiency comparison between PEMax and L-PGI. B Comparison of engineered nCas9-RT variant controls with published mRNA sequences for placement of 38 bp Bxb1 attachment site (attB) in F9 intron 1 via twin PE mechanism using paired guides with 20 overlap in PHH assayed by digital droplet PCR (ddPCR). Unpaired T test and ordinary one-way ANOVA with Gaussian distribution performed, respectively. n = 2 for PE6 test condition only. C Efficiency and fidelity of 2 nt substitution of UGC to UAU to install C282Y with silent mutation in HFE assayed by NGS. L-PGI is compared to PE-TB2 (nCas9 fused to engineered RT). Ordinary one-way ANOVA performed for efficiency to calculate p values, ns indicates not significant. n = 2 for L-PGI test condition only. D Schematic map showing location of guide target window in a 900 bp range spanning the transcription start site (TSS) preceding exon 2 of APOA1. E Efficiency of 1–3 nt substitutions performed by either PE or L-PGI shown side by side using the same spacer sequences for either pegRNA or lmgRNA and identical ngRNA by Sanger Sequencing and inference of CRISPR edits (ICE) analysis against untreated control. Paired T test performed with estimation plot showing up to 54.3 percentage gain in efficiency with average gain of 13.1 across all tested spacers. F Edit quality comparison for subset of APOA1 spacers showing comparable fidelity across loci and methods. SP4275 PE-TB3, SP4286, and SP4275 L-PGI test conditions contain n = 2 biological replicates. All error bars represent standard deviation. Source data are provided in Source Data file. Nucleotide reagents provided in Supplementary Data 1-4.