1-3 organoids of the same batch were pooled and dissociated at multiple timepoints across the organoid developmental time course. Organoids were washed three times with HBSS without Ca2+/Mg2+ (STEMCELL technologies, 37250). Single cell suspensions were obtained by adapting the protocol described in (Cowan et al. 2020) and using a papain-based dissociation kit (Miltenyi Biotec, 130-092-628). Stem cell lines were cultured in mTeSR1 (Stem Cell Technologies, 05851) with mTeSR1 supplement (Stem Cell Technologies, 05852) or mTeSR Plus (Stem Cell Technologies, 100-0274) with mTeSR Plus (Stem Cell Technologies, 100-0275) and supplemented with penicillin/streptomycin (P/S, 1:200, Gibco, 15140122) on matrigel-coated plates (Corning, 354277). Cells were split 1-2 times per week after dissociation with EDTA in DPBS (final concentration 0.5mM) (Gibco, 12605010). Media was supplemented with Rho-associated protein kinase (ROCK) inhibitor Y-27632 (final concentration 5μM, STEMCELL, 72302) the first day after thawing. To generate retinal organoids from the B7 and IMR90 lines, 600 cells were seeded in each micro-well of an agarose chamber (MicroTissues® 3D Petri Dish® micro-mold #Z764019). 409B2-iCas9 organoids were generated with 1500 cells per micro-well and treated with BMP4 (55ng/ml, Biotechne #314-BP) on day 6 with half medium change on day 9, 12 and15 (Kuwahara et al. 2015). 8,000 cells were targeted and when the cell number was not sufficient, all cells were loaded. Single cell RNA-seq libraries were generated with the Chromium Single Cell 3' V3.1 Library & Gel Bead Kit. Single cell encapsulation and library preparation were performed according to the manufacturer's protocol. Single cell RNA-seq libraries were generated with the Chromium Single Cell 3' V3.1 Library & Gel Bead Kit. Single cell encapsulation and library preparation were performed according to the manufacturer's protocol.