Fig. 5: Nick location dictates the type of PE3 editing byproducts at ATP1A1.
From: Marker-free co-selection for successive rounds of prime editing in human cells
a Schematic representation of the complementary 5′ and 3′ single-stranded DNA overhangs generated with the PE3-T804N strategy at ATP1A1 exon 17 (PAM-In configuration) and the PE3-Q118R strategy at ATP1A1 exon 4 (PAM-Out configuration), respectively. b PCR-based genotyping of ATP1A1 exon 17 and 4 from single cell-derived MTOR-F2108L/I2017T K562 clones. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 100 µM ouabain and genomic DNA was harvested after co-selection. n = 16 single cell-derived clones from one experiment. c Same as in b with single cell-derived MTOR-F2108L/E2419K K562 clones. n = 16 single cell-derived clones from one experiment. d Schematic representation of pegRNA and nick sgRNA target sites with PAMs facing inwards (PAM-In configuration) and outwards (PAM-Out configuration) at ATP1A1 exon 4. e PCR-based genotyping of ATP1A1 exon 4 from single cell-derived K562 clones targeted with PAM-out and PAM-in configurations using different nick sgRNAs. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 0.5 µM ouabain and genomic DNA was harvested after co-selection. n = 17 single cell-derived clones for each condition from one experiment. Ins, insertion. Del, deletion.
