Fig. 5: Modified pegRNAs mitigate scaffold incorporation by PE3 and highly processive PE systems.

a Editing of FANCF with PE3 in K562 cells. Cells were electroporated with RNPs and either pegRNA or rSp-pegRNA to install indicated substitution at the FANCF genomic locus. b Quantification of data from (a). “Prime edits containing scaffold” was calculated as total number of amplicon-seq prime edited reads with scaffold integration per total prime edited reads. c Editing with prime editing nucleases PEn6d and PEn** in HEK293T cells. Cells were transfected with plasmids expressing PEn6d or PEn** in combination with synthetic pegRNA or C96Me-pegRNA to install a 7 bp insertion (ins.) at the VEGFA genomic locus. d Quantification of data from (c). “Prime edits containing scaffold” was calculated as total number of amplicon-seq prime edited reads with scaffold integration per total prime edited reads. e Editing with prime editing nickases PE6d and PE** in HEK293T cells. Cells were transfected with plasmids expressing PE6d or PE** in combination with synthetic pegRNA or C96Me-pegRNA to install a 7 bp insertion (ins.) at the VEGFA genomic locus. Editing outcomes were analyzed by amplicon-seq and quantified using CRISPResso2 in the prime editing mode. f Quantification of data from (e). “Prime edits containing scaffold” was calculated as total number of amplicon-seq prime edited reads with scaffold integration per total prime edited reads. Editing outcomes were analyzed by amplicon-seq and quantified using CRISPResso2 in the prime editing mode. Plots show mean ± SD of n  =  3 biological replicates. Prime edits precise = intended prime edits; Prime edits all = precise prime edits + prime edits co-occurring with indels; Scaffold incorporated = prime edits with at least one additional nucleotide matching scaffold; Indels = non-prime edited insertions or deletions. Source data are provided as a Source Data file.