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pubmed:abstractText |
Dominant mutations in the rhodopsin gene, which is expressed in rod photoreceptor cells, are a major cause of the hereditary-blinding disease, autosomal dominant retinitis pigmentosa. Therapeutic strategies designed to edit such mutations will likely depend on the introduction of double-strand breaks and their subsequent repair by homologous recombination or non-homologous end joining. At present, the break repair capabilities of mature neurons, in general, and rod cells, in particular, are undefined. To detect break repair, we generated mice that carry a modified human rhodopsin-GFP fusion gene at the normal mouse rhodopsin locus. The rhodopsin-GFP gene carries tandem copies of exon 2, with an ISceI recognition site situated between them. An ISceI-induced break can be repaired either by non-homologous end joining or by recombination between the duplicated segments, generating a functional rhodopsin-GFP gene. We introduced breaks using recombinant adeno-associated virus to transduce the gene encoding ISceI nuclease. We found that virtually 100% of transduced rod cells were mutated at the ISceI site, with ?85% of the genomes altered by end joining and ?15% by the single-strand annealing pathway of homologous recombination. These studies establish that the genomes of terminally differentiated rod cells can be efficiently edited in living organisms.
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