title: Efficient mutagenesis of the rhodopsin gene in rod photoreceptor neurons in mice.
abstract: 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.
authors.affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Fung
authors.initials: F
authors.lastname: Chan
authors.forename: William W
authors.initials: WW
authors.lastname: Hauswirth
authors.forename: Theodore G
authors.initials: TG
authors.lastname: Wensel
authors.forename: John H
authors.initials: JH
authors.lastname: Wilson
mesh_terms: D000818:Animals; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D000229:Dependovirus / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D055879:Gene Knock-In Techniques; D005822:Genetic Vectors / Q000008:administration & dosage; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007267:Injections; D051379:Mice; D023421:Models, Animal; D016296:Mutagenesis; D009154:Mutation; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism*; D012243:Rhodopsin / Q000235:genetics*
chemical_list: D049452:Green Fluorescent Proteins; D012243:Rhodopsin; D004706:Endodeoxyribonucleases