Lin Yuan1, Haobin Yao1, Yuxin Xu1, Mao Chen1, Jichao Deng1, Yuning Song1, Tingting Sui1, Yong Wang1, Yongye Huang2, Zhanjun Li1, Liangxue Lai3. 1. Jilin Provincial Key Laboratory of Animal Embryo Engineering, Institute of Zoonosis, College of Animal Science, Jilin University, Changchun, China. 2. Jilin Provincial Key Laboratory of Animal Embryo Engineering, Institute of Zoonosis, College of Animal Science, Jilin University, Changchun, China 2College of Life Science and Health, Northeastern University, Shenyang, China. 3. Jilin Provincial Key Laboratory of Animal Embryo Engineering, Institute of Zoonosis, College of Animal Science, Jilin University, Changchun, China 3CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
Abstract
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.
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.