Fig. 6: Assessing the impact of complementary-strand nick locations on prime editing outcomes.

a Schematic representation of the EBFP to EGFP reporter system using different nick sgRNAs with PAMs facing outwards (PAM-Out) or inwards (PAM-In) towards the pegRNA. b Schematic of the FACS-based quantification of EBFP to EGFP conversion via PE. In K562 cells homozygous for EBFP integration at ATP1A1 (triallelic), FACS-based analysis reports PE outcomes indirectly; (i) EBFP(+)/EGFP(+) cells result from monoallelic or biallelic PE, (ii) EBFP(−)/EGFP(+) cells are generated by either triallelic PE or combinations of mono- and biallelic PE along with indels*, (iii) EBFP(−)/EGFP(−) occur from indels on the three copies of the reporter. Indels are defined broadly in this context as any edits that inactivates the reporter. c FACS-based quantification of EBFP to EGFP conversion via PE after co-selection. K562 cells stably expressing the EBFP reporter from the ATP1A1 locus were transfected with PE3max vectors targeting ATP1A1 exon 4 (pegRNA-Q118R_v1) and EBFP (epegRNA). Cells were treated with 100 µM ouabain starting 3 days post-transfection until all non-resistant cells were eliminated. n = 3 independent biological replicates performed at different times with equivalent results (see Supplementary Figs. 22,23). d PCR-based genotyping of EGFP(+) single cell-derived K562 clones targeted with the PE2 or PE3b strategy. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 100 µM ouabain and genomic DNA from EGFP(+) clones was harvested after co-selection. The number of precise prime edited alleles was determined from BEAT Sanger sequencing trace analysis and small indels were analysed with DECODR. n = 17 single cell-derived clones for each condition from one experiment. e PCR-based genotyping of EBFP from single K562 cell-derived clones after sorting for EBFP(−)/EGFP(+) cells. The number of precise prime edited alleles was determined from BEAT Sanger sequencing trace analysis and small indels were analysed with DECODR. Larger insertions and deletions are indicated, and homozygous single cell-derived clones are highlighted in bold and green. PE alleles harboring pegRNA scaffold incorporation are indicated (si). n = 17 and 16 single cell-derived clones from one experiment for the PE3-G3 and PE3-G6 conditions, respectively. Ins, insertion. Del, deletion.