Efficient mutagenesis of the rhodopsin gene in rod photoreceptor neurons in mice

Nucleic Acids Res. 2011 Aug;39(14):5955-66. doi: 10.1093/nar/gkr196. Epub 2011 Apr 7.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • DNA Breaks, Double-Stranded*
  • DNA Repair*
  • Dependovirus / genetics
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism
  • Gene Knock-In Techniques
  • Genetic Vectors / administration & dosage
  • Green Fluorescent Proteins / genetics
  • Humans
  • Injections
  • Mice
  • Models, Animal
  • Mutagenesis
  • Mutation*
  • Retinal Rod Photoreceptor Cells / metabolism*
  • Rhodopsin / genetics*


  • Green Fluorescent Proteins
  • Rhodopsin
  • Endodeoxyribonucleases