Construction And Characterization Of Congenital Cataracts Mediated By CRISPR/Cas9 In Rabbits

Congenital cataracts are the main cause of blindness in children. According to clinical data, about a third of the congenital cataracts are taken as genetic disease and indicated as autosomal dominant inheritance. The abnormal expression of Crystallin and Connexin protein is able to cause cataracts in the current researches. Therefore, CRYAA and GJA8 geneswere selected as the target genes and their roles in cataracts were studiedin our study. The αA-Crystallin protein encoded by CRYAA gene accounts for 40% of the total proteins in lens, plays an important role in preventing the false aggregation of proteins. Connexin 50 protein encoded by GJA8 gene takes part in the formation of gap junction channel, making metabolites and water exchanging among fiber cells, thus maintaining the transparency of lens. Different mutations may cause different symptoms of cataracts, and have different molecular and cellular pathogenesis mechanism. Therefore, uncovering the underlying mechanism of cataract pathogenesis is very important for its treatment. Given the similarity in the eye size between rabbits and human beings, the rabbits were chosen as the human eyes’ disease model in this study.Recently, CRISPR/Cas9 system, an immune defense system, has been used extensively. CRISPR/Cas9 technology has become the first choice in modifying genome due to its advantages, which includeshort design cycle, low cost and high efficiency in editing genes.According to the genome database in NCBI and Ensemble,two sg RNAs loci were designed to target coding regions of CRYAA and GJA8 genes.It is successful to microinject the Cas9 and sg RNA m RNAs into the zygotes.The methods of PCR, HE staining, western blotting, immunofluorescence and TUNEL assays were used to analyze the genotype and phenotypes in F0 generation rabbits.CRYAA+/- rabbits represented lens opacity, microphthalmia with small lenses and congenital cataracts. The CRYAA protein was decreased significantly in immunohistochemistry assay. Furthermore, the results of TUNEL assay demonstrated that the CRYAA mutation may induce cell apoptosis in the lens epithelium and cornea, which compared to the WT rabbits.The validated genetic and histology results suggested that GJA8+/- rabbits presented lens opacity, microphthalmia with small lenses and congenital cataracts.The GJA8 protein was decreased significantly in immunolfuorescence assay. Furthermore, TEM results confirmed the much shorter gap junction in the cortical fibers of GJA8 mutant rabbit, compared to the WT rabbits. F1 generation rabbits had been acquired via intercrossing between the F0 rabbits, these data revealed the genotypes and disrupted function of CRYAA and GJA8 were inheritable in our cataract rabbit models.In conclusion, there were typical syptoms of congenital cataracts in rabbits, which were modified in CRYAA and GJA8 genes. This novel cataract rabbit may provide more reliable animal models for CRYAA and GJA8 genes’ function researching, drug screening and cataract prevention.