Journal
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
Volume 55, Issue 1, Pages 387-395Publisher
ASSOC RESEARCH VISION OPHTHALMOLOGY INC
DOI: 10.1167/iovs.13-13278
Keywords
genetic engineering; TALEN; CRB1 associated disease; KOMP; gene therapy
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Funding
- National Institutes of Health [OD011190, EY011996]
- National Institutes of Health Research Fellowship under Ruth L. Kirschstein National Research Service Award
- National Institutes of Health Cancer Center Support (CORE) [5P30CA034196]
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PURPOSE. We directly corrected the mouse Crb1(rd8) gene mutation, which is present in many inbred laboratory strains derived from C57BL/6N and complicates genetic studies of retinal disease in mice. METHODS. Fertilized C57BL/6NJ oocytes were coinjected with mRNAs encoding a transcription activator-like effector nuclease (TALEN) targeting the Crb1(rd8) allele plus single-stranded oligonucleotides to correct the allele. The oligonucleotides included additional nucleotide changes to distinguish the corrected allele (Crb1(em1Mvw)) from wildtype Crb1 and to minimize TALEN recutting. Oligonucleotide length, concentration of injected oligonucleotides and TALEN mRNAs were varied to optimize homology-directed repair of the locus. Following microinjection, embryos were carried to term in pseudopregnant females. Correction efficiency was assessed by PCR analysis of the Crb1(em1Mvw) allele. Phenotypic correction was demonstrated by fundus imaging and optical coherence tomography of live mice, and by confocal fluorescence microscopy of retinal flat mounts. RESULTS. Under optimal conditions, homology-directed repair was observed in 27% (8/30) of live-born animals and showed minimal illegitimate recombination of donor DNA. However, extensive founder mosaicism was evident, emphasizing the need to analyze offspring of founder animals. Unlike C57BL/6NJ mice, which exhibited external limiting membrane fragmentation and regional retinal dysplasia, heterozygous Crb1(em1Mvw)/Crb1(rd8) mice showed a normal retinal phenotype. CONCLUSIONS. The C57BL/6NJ-Crb1(rd8) mutation and its associated retinal phenotypes were corrected efficiently by TALEN-mediated homology-directed repair. The C57BL/6NJCrb1(em1Mvw) mice generated by this strategy will enhance ocular phenotyping efforts based on the C57BL/6N background, such as those implemented by the International Mouse Phenotyping Consortium (IMPC) project.
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