Extended Data Fig. 4: Generation of the ABCA4G1961E human mutant iPSC line.
From: High-efficiency base editing in the retina in primates and human tissues
A) Strategy for the generation of an ABCA4G1961E iPSC line. ABCA4 exon 42 (blue box) with flanking introns (top). The PAM site is highlighted in purple, and the gRNA binding site is indicated by a black dashed line. The black arrowheads point to the PAM disruption site (silent mutation) and the red arrowheads point to the ABCA4 c.5882G>A mutation. Representative Sanger sequencing trace of the ABCA41961E/E clone that was selected for human retinal organoid induction (bottom). B) Results from targeted deep-sequencing of the ABCA41961G/E (top) and ABCA41961E/E (bottom) clone confirming successful knock-in of the target mutation in a heterozygous or homozygous form. C) Results from the iPSC digital aneuploidy test, confirming the genomic integrity of the ABCA41961G/E (top) and ABCA41961E/E (bottom) clone. These clones were used for human retinal organoid induction. D) Confocal images of ABCA41961G/E (left) and ABCA41961E/E (right) iPSCs. Green: antibody for pluripotency markers (NANOG, SOX2, OCT4, SSEA4); grey: Hoechst (scale bars: 100 µm). The experiment was performed once with one biological replicate.
