Purpose: Although progresses have been made in the understanding of the genetic basis for Retinitis Pigmentosa (RP), the development of therapeutic intervention is still lagging behind. Gene therapy was successfully applied to retina degeneration but only to recessive mutations. Rhodopsin (RHO) mutations represent a common cause of RP, accounting for 25% of adRP and 8 to 10% of all RP. We aimed at developing genome editing tools to knock out the RHO defective alleles by introducing a double strand break (DSB) into the target gene. Methods: The CRISPR/Cas9 system was developed to target the first exon of the human RHO gene by designing 2 gRNAs. The system was tested in vitro in HeLa clones expressing RHO. The effects on the RHO gene were evaluated by sequencing and by analyses at mRNA and protein levels. We expressed by electroporation these 2 gRNAs and Cas9 in vivo in transgenic mice expressing human RHO with P23H mutation. Genome editing was evaluated by sequencing genomic DNA from targeted cells and by analyzing RHO mRNA and protein. Results: Two gRNAs were designed in the first exon of the RHO gene and one of them targeted the P23H mutation. The two gRNAs were tested singularly or together in vitro on HeLa clones stably expressing RHO. We demonstrated insertions or deletions (indels) in the genomic DNA specifically in the RHO gene. Indels caused strong reduction of the RHO mRNA and of RHO protein up to 90%. The higher effect was obtained with the two gRNAs together. The two gRNAs were then in vivo expressed with Cas9 in photoreceptors of transgenic mice by electroporation. Targeted cells were tracked by co-expression with EGFP. EGFP+ cells were FACS sorted and indels in the human P23H RHO gene were analyzed by sequencing. We were able to detect up to 30% of genome editing in vivo. We also detected reduction of human RHO mRNA expression as well as RHO protein. Conclusions: We developed new tools to downregulate mutant RHO in dominant forms of RP. The CRISPR/Cas9 system reveled a high efficiency and should be tested for knock-down followed by gene replacement approaches.
In vitro and in vivo genome editing of the RHO gene to downregulate dominant mutations / Marigo, Valeria; Latella, Maria Carmela; DI SALVO, MARIA TERESA; Recchia, Alessandra. - In: INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE. - ISSN 0146-0404. - 57:12(2016).