期刊
NUCLEIC ACIDS RESEARCH
卷 39, 期 14, 页码 5955-5966出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkr196
关键词
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资金
- National Institutes of Health [EY11731, EY07981, EY13729, EY11123, EY08571]
- Vision Research Core Grant [EY002520]
- Robert A. Welch Foundation
- Macular Vision Research Foundation
- Research to Prevent Blindness, Inc.
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 similar to 85% of the genomes altered by end joining and similar to 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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