Fig. 7: Therapeutic base editing by en31-ABEmax8.17d in a leber congenital amaurosis 2 patient- specific iPSC line (LCA2−2).
From: PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics
a Representative image showing iPSCs reprogrammed from the skin explant-derived human dermal fibroblasts (HDF) of an LCA2 patient (n = 1, P = passage number). A total of 12 clones were picked at P0 and clonally expanded. A stable patient-specific iPSC line (LCA2−2) at passage 12 was used for editing studies. b Schematic showing patient-specific point mutation correction by en31-ABEmax8.17d, targeting a premature stop codon in exon 9 of RPE65 gene. c Ballon plot showing the A to G editing events (%) as obtained from amplicon sequencing upon targeting RPE65-E9 with en31-ABEmax8.17d constructs encoding sgRNAs containing either 20- or 21-nt spacer sequences. The targeted PAM sequences, 5’-AGG-3’, 5’-GGA-3’ and 5’-AAG-3’ are underlined. Mean of n = 3 independent biological replicates are shown. d Bar plot showing the efficiency of A to G editing (%) plotted on the Y-axis, as obtained from amplicon sequencing of two en31-ABEmax8.17d-treated clonal lines, LCA2−2-BE1 (in red, clone 1), LCA2−2-BE2 (in blue, clone 2). Error bars represent mean ± SEM of n = 3 independent sequencing replicates with individual values shown as dots. c, d Target bases are shown on the x-axis, and counted w.r.t. g21 and 5’-AGG-3’ PAM. The target sequence is shown and the pathogenic-mutation (A8) is highlighted in red. e Representative phase contrast images showing the lower magnification view of the cellular morphology and levels of pigmentation in iPSC-derived RPE cultures (n = 3) at days 45 (upper panels) and at day 75 (lower panels) of differentiation. f Representative immunoassayed iPSC-RPE culture images (n = 2), showing their morphology and expression of RPE-specific markers such as, Phalloidin-FITC (green), PI (red) (i-iii); MITF (green), PI (red) (iv-vi); and RPE65 (green), DAPI (blue) (vii-ix). g Representative western blot images showing the expression of different RPE-specific proteins in iPSC-derived RPE cell lysates of healthy control (F2), patient-specific (LCA2−2) and mutation-corrected (LCA2−2-BE1) lines (n = 2). β-ACTIN served as loading control. Asterisk (*) marks a non-specific band. Arrowhead marks the truncated RPE65 protein band in LCA2−2-RPE lysate. Arrow marks a minor isoform-specific band of PAX6. a, e, f Scale bars are as indicated. Raw data are provided in Source Data file.
