Engineered zinc finger nuclease-mediated homologous recombination of the human rhodopsin gene.
OBJECTIVE
Novel zinc finger nucleases (ZFNs) were designed to target the human rhodopsin gene and induce homologous recombination of a donor DNA fragment.


METHODS
Three-finger zinc finger nucleases were designed based on previously published guidelines. To assay for ZFN specificity, the authors generated human embryonic retinoblast cell lines stably expressing a Pro23His rhodopsin, the most common mutation associated with autosomal dominant retinitis pigmentosa in North America. They report quantification of these rhodopsin-specific ZFNs to induce a targeted double-strand break in the human genome, demonstrate their ability to induce homologous recombination of a donor DNA fragment, and report the quantification of the frequency of ZFN-mediated homologous recombination.


RESULTS
Compared with endogenous homologous recombination, the authors observed a 12-fold increase in homologous recombination and an absolute frequency of ZFN-directed homologous recombination as high as 17% in the human rhodopsin gene.


CONCLUSIONS
ZFNs are chimeric proteins with significant potential for the treatment of inherited diseases. In this study, the authors report the design of novel ZFNs targeting the human rhodopsin gene. These ZFNs may be useful for the treatment of retinal diseases such as retinitis pigmentosa, one of the most common causes of inherited blindness in the developed world. Herein, they also report on several aspects of donor fragment design and in vitro conditions that facilitate ZFN-mediated homologous recombination.
Greenwald
David L
DL
Department of Ophthalmology, Tufts University School of Medicine, Boston, MA 02111, USA.
Cashman
Siobhan M
SM
Kumar-Singh
Rajendra
R
D015153:Blotting, Western; D002460:Cell Line; D004706:Endodeoxyribonucleases / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D005818:Genetic Engineering; D006801:Humans; D016297:Mutagenesis, Site-Directed; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D011995:Recombination, Genetic; D012160:Retina / Q000196:embryology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012243:Rhodopsin / Q000235:genetics*; D014162:Transfection; D016335:Zinc Fingers / Q000235:genetics*
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012243:Rhodopsin; D004706:Endodeoxyribonucleases