Extended Data Fig. 2: In vitro validation of editing strategy in primary patient fibroblasts.
From: Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10
a, Targeted deletion at the CEP290 locus in primary patient fibroblasts transfected with plasmids encoding SaCas9 and seven different pairs of CEP290 gRNAs, as quantified by Droplet Digital PCRR. Left, cell line IVS26#36, n = 4 independent transfections performed on different days, error bars represent standard deviation. Right, cell line IVS26#35, n = 1. b, Quantification of WT 26-27 (blue) and mutant 26-X-27 (orange) CEP290 mRNA transcripts in IVS26#35 primary patient fibroblasts by qRT–PCR. CEP290 expression is normalized to beta-actin. n = 2 independent transfections performed on different days, qRT–PCR run in triplicate. Line represents mean. c, representative western blot from IVS26 primary patient fibroblasts transfected with plasmids encoding SaCas9 and seven different pairs of CEP290 gRNAs. Experiment was performed twice in each cell line with similar results. d, Quantification of CEP290 full-length protein expression in IVS26#36 and -#35 cell lines transfected with plasmids encoding SaCas9 and seven different pairs of CEP290 gRNAs. Expression level fold change over control calculated by densitometry of western blot bands and normalized to control. n = 4 biological replicates (two different transfections on different days, each in the two different cell lines), error bars represent mean and standard deviation.
