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Journal Article
Research Support, Non-U.S. Gov't
CRISPR/Cas9-Mediated Mutation of αA-Crystallin Gene Induces Congenital Cataracts in Rabbits.
Investigative Ophthalmology & Visual Science 2017 May 2
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.
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.
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