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CRISPR/Cas9 Gene Editing

Restoration of RPGR expression in vivo using CRISPR/Cas9 gene editing


Mutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) cause the X-linked form of inherited retinal degeneration, and the majority are frameshift mutations in a highly repetitive, purine-rich region of RPGR known as the OFR15 exon. Truncation of the reading frame in this terminal exon ablates the functionally important C-terminal domain. We hypothesized that targeted excision in ORF15 by CRISPR/Cas9 and the ensuing repair by non-homologous end joining could restore RPGR reading frame in a portion of mutant photoreceptors thereby correcting gene function in vivo. We tested this hypothesis in the rd9 mouse, a naturally occurring mutant line that carries a frameshift mutation in RPGRORF15, through a combination of germline and somatic gene therapy approaches. In germline gene-edited rd9 mice, probing with RPGR domain-specific antibodies demonstrated expression of full length RPGRORF15 protein. Hallmark features of RPGR mutation-associated early disease phenotypes, such as mislocalization of cone opsins, were no longer present. Subretinal injections of the same guide RNA (sgRNA) carried in AAV sgRNA and SpCas9 expression vectors restored reading frame of RPGRORF15 in a subpopulation of cells with broad distribution throughout the retina, confirming successful correction of the mutation. These data suggest that a simplified form of genome editing mediated by CRISPR, as described here, could be further developed to repair RPGRORF15 mutations in vivo.


Retinitis pigmentosa (RP) is a heterogenous group of inherited retinal diseases caused by a progressive degeneration of rod and cone photoreceptors that results in the eventual loss of vision. Mutations in more than 200 genes cause RP and collectively these affect one in 3000–5000 people, representing a major cause of inherited forms of blindness globally. Mutations causing X-linked RP (XLRP) are particularly severe with retinal disease presenting within the first few decades of life. The majority of mutations causing XLRP have been identified as loss-of-function alleles in the gene encoding retinitis pigmentosa GTPase regulator (RPGR).

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