Fig. 5

Single nickase-mediated gene editing results in c.217 C clone for CEP disease modeling a (Left) Scheme of gene editing approach to convert wild-type HEK293T (WT HEK) into homozygous c.217 C HEK clone using nickase and a 181nt-ssODN carrying c.217 C mutation (called 181nt-ssODN-c.217 C). (Right) Detailed view of exon 4 region and CRISPR-mediated HDR design using a c.217T-targeting sgRNA and a 181nt-ssODN-c.217 C carrying c.217 C mutation (red) in addition to silent SacI restriction site (blue). Expected cleavage position using nickase is indicated with a red arrow. b–d (From left to right) Illustrative flow cytometry results for fluorocyte analysis, representative RFLP analysis, sequence spanning UROS exon 4 c.217 position obtained by Sanger sequencing and indels and HDR quantification by TIDER analysis (b) for WT HEK, (c) for cells transfected with nickase and a 181nt-ssODN-c.217 C (Mixed HEK population) and (d) for sorted and subcloned fluorocytes (PE-Cy5A-positive), called c.217 C HEK clone. Loq: limit of quantification. e Characterization of c.217 C HEK clone. UROS functionality assay with (Left) quantification of UROS-specific activity and (Right) fluorocyte frequencies from WT HEK or c.217 C HEK clone. Values for UROS-specific activity are normalized against WT HEK. Results are presented as mean ± SEM. For (e), source data are provided as a Source data file