Fig. 3: PRINS editing with modified pegRNAs in primary human hepatocytes and mouse embryos.

a PRINS editing at PCSK9 and HBEGF genomic loci in primary human hepatocytes transfected with PEn mRNA and a synthetic pegRNA or rSp-pegRNA to install small insertions (ins.). Editing outcomes were analyzed by amplicon-seq and CRISPResso2 in the prime editing mode. “Precision score” was calculated as total number of amplicon-seq reads with precise prime edit per overall editing. “Prime edits with scaffold” was calculated as total number of amplicon-seq prime edited reads with scaffold integration per total prime edited reads. Plots show mean ± SD of n  =  3 biological replicates. Statistical difference was determined using Student’s t test (paired, two-tailed). Prime edits precise = intended prime edits; Prime edits all (no scaffold) = precise prime edits + prime edits co-occurring with indels not derived from scaffold; Scaffold incorporated = prime edits with at least one additional nucleotide derived from scaffold; Indels = non-prime edited insertions or deletions; *P  <  0.05, **P  <  0.01, ***P  <  0.001. Calculated P values: PCSK9 “Prime edits - precise” = 0.005, PCSK9 “Prime edits with scaffold” = 0.0057, PCSK9 “Precision score” = 0.0104, HBEGF “Prime edits - precise” = 0.0123, HBEGF “Prime edits with scaffold” = 0.0018 HBEGF “Precision score” = 0.0027. b PRINS editing of mouse embryos using electroporation of PEn-pegRNA or PEn-rSp-pegRNA RNP complexes to install a 5 bp insertion into the endogenous Map3k15 locus. Each data point represents a single embryo edited with either pegRNA (n = 25) or rSp-pegRNA (n = 24). Single embryos were analyzed by amplicon-seq and CRISPResso2 after 5 days of in vitro cultivation. “Prime edits with scaffold” was calculated as total number of amplicon-seq prime edited reads with scaffold integration per total prime edited reads. “Precision score” was calculated as total number of amplicon-seq reads with precise prime edit per overall editing. “Reads with precise edit” corresponds to a total number of precisely edited amplicon-seq reads in each embryo. Error bars represent mean ± SD. Statistical significance was determined using Student’s t test (unpaired, two-tailed). *P  <  0.05, **P  <  0.01, ***P  <  0.001. Calculated P values: “Prime edits with scaffold” <0.0001, “Precision score” = 0.011, “Reads with precise edit” = 0.0319. Source data are provided as a Source Data file.