In vitro and in vivo CRISPR/Cas9-mediated genome editing to downregulate dominant mutations in Rhodopsin gene

Many progresses have been made in understanding the genetic basis for Retinitis Pigmentosa (RP), however therapeutic interventions are still lagging behind. Rhodopsin (RHO) mutations represent a common cause of RP, accounting for 25% of autosomal dominant RP and 8-10% of all RP (Hartong et al., 2006) with more than 100 different mutations identified so far. Here we show the application of CRISPR-Cas9 technology to knock out the RHO defective alleles by introducing double strand breaks into the target gene. We designed single or double gRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. The two gRNAs were tested singularly or together in vitro in HeLa clones stably expressing P23H RHO. Cel I assay, TIDE and sequencing analyses demonstrated insertions or deletions (indels) in the genomic DNA specifically in the RHO gene, which caused strong reduction of RHO expression up to 90%. The higher effect was obtained with two gRNAs together. The CRISPR/Cas9 plasmid expressing two gRNAs were then in vivo tested in P23H RHO transgenic mice by sub-retinal electroporation, together with EGFP expressing plasmids. Analysis of indels in FACS-sorted EGFP+ cells demonstrated up to 30% of in vivo genome editing of the human P23H RHO gene, without targeting of the murine Rho allele. We also detected reduction of RHO at mRNA and protein levels. Thus, successful in vivo application of the CRISPR/Cas9 system confirms its efficacy as a genetic engineering tool and its potential use in gene therapy.