title: Correction of the Crb1rd8 allele and retinal phenotype in C57BL/6N mice via TALEN-mediated homology-directed repair.
abstract: OBJECTIVE
  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 wild-type 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/6NJ-Crb1(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.
authors.affiliation: The Jackson Laboratory, Bar Harbor, Maine.
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Low
authors.forename: Mark P
authors.initials: MP
authors.lastname: Krebs
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
authors.forename: Shengdar Q
authors.initials: SQ
authors.lastname: Tsai
authors.forename: Patsy M
authors.initials: PM
authors.lastname: Nishina
authors.forename: Michael V
authors.initials: MV
authors.lastname: Wiles
mesh_terms: D000483:Alleles; D000818:Animals; D004195:Disease Models, Animal; D053595:Embryonic Stem Cells / Q000378:metabolism; D005136:Eye Proteins; D005260:Female; D015316:Genetic Therapy / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL / Q000235:genetics*; D018613:Microscopy, Confocal; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D016133:Polymerase Chain Reaction; D059767:Recombinational DNA Repair / Q000235:genetics*; D012160:Retina / Q000378:metabolism* / Q000473:pathology; D012162:Retinal Degeneration / Q000235:genetics* / Q000378:metabolism / Q000628:therapy; D041623:Tomography, Optical Coherence
chemical_list: C453730:Crb1 protein, mouse; D005136:Eye Proteins; D009419:Nerve Tissue Proteins
keywords: CRB1 associated disease; KOMP; TALEN; gene therapy; genetic engineering