Establishment and rescue of fibroblast cell lines carrying a nonsense mutation of RB1 by CRISPR-based base editing.

Pathogenic variants of the RB1 gene have commonly been found in many cancer types, including retinoblastoma. Nonsense mutations are the most common mutation type in retinoblastoma; however, few cell lines mimic nonsense mutations in the RB1 gene that are commonly observed in patients. Here, we established retinoblastoma-like cell lines carrying mono- and bi-allelic nonsense mutations in the RB1 gene. We introduced the R552X mutation using target activation-induced cytidine deaminase base editing and successfully constructed cell lines carrying mono- and bi-allelic mutations. The model cell lines showed decreased RB1 expression at both the mRNA and protein levels, and increased cell proliferation. Furthermore, we rescued the nonsense mutation in the RB1 gene in model cell lines by converting stop codon 552 to tryptophan using an adenine base editor. This approach may be applicable for establishing cell lines with pathogenic variants found in patients and suggests a strong potential for the application of gene editing as a therapeutic strategy.