Fig. 2

Generation of LCA5 KO and isogenic control iPSCs and differentiation to retinal organoids. A) Sanger sequence trace of LCA5 KO iPSC (LCA5 KO1) showing a 2-bp deletion in exon 3 of LCA5 gene generated by CRISPR/Cas9 and NHEJ gene editing. B) Bright-field images of iPSC-derived LCA5 KO and isogenic control retinal organoids at D120, D150 and D180 of retinal development. Inset boxes showing the development of photoreceptor brush bor­ders which start to emerge at D180. Scale bars 250 μm. C) RT-PCR of isogenic control and LCA5 KO iPSC and retinal organoids (n = 2 per condition from one differentiation) at D120, D150 and D180 for retinal differentiation markers ARR3, CRX, NRL, CHX10, NR2E3, PAX6, REEP6.1 (upper band), REEP6.2 (lower band). GAPDH was used as a reference transcript. D) Western blot of control, LCA5 KO (KO1 and KO2) and LCA5 JB342 patient retinal organoids at D150 showing successful knockdown of LCA5 protein. Recoverin (RCVRN) was used as a photoreceptor-specific marker and GAPDH as a loading control. Results are from pooling together n = 3 retinal organoids per condition from two differentiations per line