Fig. 1

Generation of the RB1 gene-mutated human fibroblast cell lines using the cytosine base editor (CBE). (A) Schematic diagram displaying the induction of the retinoblastoma-causing mutation, R552X, in RB1 exon 17. The protospacer adjacent motif, Target-AID editing window, and targeted cytosine are highlighted in blue, green, and red, respectively. This type of retinoblastoma occurs when arginine 552 changes to a stop codon. (B) Schematic diagram of the CBE, Target-AID. A nickase Cas9 (D10A)-sgRNA RNP complex forms an R-loop at the target site. The fused cytidine deaminase, PmCDA1, converts an exposed cytidine into a thymidine. (C) The procedure of base editing and sequential screening with high-throughput sequencing for the establishment of homozygous and heterozygous RB1-mutated cell lines are shown. (D) Edited frequencies (%) of bulk, first, and second screening populations, as measured by next-generation sequencing (NGS). Green dots represent bulk cell populations which were used for first screening. Blue and red dots represent cell populations used in the second screening process for heterozygous and homozygous cell lines, respectively (mean ± SEM). (E) Edited frequencies (%) of established R552X homozygous and heterozygous cell lines measured by NGS.