CRISPR/Cas9-Mediated Mutation of αA-Crystallin Gene Induces Congenital Cataracts in Rabbits

Invest Ophthalmol Vis Sci. 2017 May 1;58(6):BIO34-BIO41. doi: 10.1167/iovs.16-21287.


Purpose: The present study aimed to investigate the role of the αA-crystallin gene in inducing congenital cataracts in rabbits and to construct a novel animal model for characterization and pathologic analysis of congenital cataracts for future research.

Methods: We generated αA-crystallin gene knockout rabbits with congenital cataracts by coinjection of Cas9 mRNA and sgRNA into zygotes. Cataract phenotypes were investigated in a repeated study of 19 F0-generation and 11 F1-generation rabbits with αA-crystallin gene mutations. Heritability was analyzed by PCR, sequencing, slim lamp, hematoxylin eosin staining, immunohistochemistry, and Western blot.

Results: We found αA-crystallin gene mutations in all 19 F0-generation pups (100%) with indel mutations in the αA-crystallin gene ranging from 3 to 52 bp. Off-target assay revealed that none of the potential off-target sites exhibited mutations, demonstrating that off-target mutagenesis was not induced by cytoplasmic microinjection of in vitro-transcribed Cas9 mRNA. Slim lamp assay revealed that 15 of 19 live pups (78.9%) exhibited typical phenotypes, including congenital cataracts, microphthalmia, obscurity, and early atrophy of the lens, and failed differentiation of lens fibers. Histologic hematoxylin and eosin staining showed that αA-crystallin gene knockout rabbits exhibited smaller lenses. Production of the αA-crystallin protein was determined to be dramatically reduced in αA-crystallin gene knockout rabbits. We induced αA-crystallin gene mutations and phenotypes in F1-generation rabbits.

Conclusions: Our data suggest that CRISPR/Cas9-mediated mutation of the αA-crystallin gene in rabbits recapitulates phenotypes of congenital cataracts, microphthalmia, obscurity, and early atrophy of the lens, and failed differentiation of lens fibers. These findings suggest the possibility of a new animal model of congenital cataracts, which should be used to further investigate the association between mutations in αA-crystallin gene and congenital cataracts in humans.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Cataract / congenital
  • Cataract / genetics*
  • Cataract / metabolism
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • DNA / genetics*
  • DNA Mutational Analysis
  • Disease Models, Animal
  • Immunohistochemistry
  • Lens, Crystalline / metabolism*
  • Mutation*
  • Phenotype
  • Polymerase Chain Reaction
  • Rabbits
  • alpha-Crystallin A Chain / genetics*
  • alpha-Crystallin A Chain / metabolism


  • alpha-Crystallin A Chain
  • DNA