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. 2022 Oct 7;13:5909. doi: 10.1038/s41467-022-33669-z

Fig. 2. Robust co-selection for successive rounds of prime editing.

Fig. 2

a Schematic of the strategy for performing successive rounds of co-selection. Cells harboring modifications at ATP1A1 (T804N) and GOI A are first co-selected with 0.5 µM ouabain. Following the first round, a subsequent round of co-selection occurs at GOI B via modification at ATP1A1 (Q118R) with 100 µM ouabain. b PE and small indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells harboring the ATP1A1-T804N and RUNX1 + 1 ATG insertion (GOI A) modifications (Fig. 1c) were transfected with PE3 vectors targeting ATP1A1 exon 4 (Q118R) and the indicated GOI B. Genomic DNA was harvested 3 days post-transfection (before selection) and cells were treated (ouabain) or not (untreated) with 100 µM ouabain until all non-resistant cells were eliminated. c Same as in b with K562 cells harboring the ATP1A1-T804N and EMX1 + 1 G to C (GOI A) modifications (See Fig. 1c). n = 2 independent biological replicates performed at different times. Source data are provided in the Source Data file.