Abstract
CRISPR/Cas9 has recently been developed as an efficient genome engineering tool. The rabbit is a suitable animal model for studies of metabolic diseases. In this study, we generated ATP7B site-directed point mutation rabbits to simulate a major mutation type in Asians (p. Arg778Leu) with Wilson disease (WD) by using the CRISPR/Cas9 system combined with single-strand DNA oligonucleotides (ssODNs). The efficiency of the precision point mutation was 52.94% when zygotes were injected 14 hours after HCG treatment and was significantly higher than that of zygotes injected 19 hours after HCG treatment (14.29%). The rabbits carrying the allele with mutant ATP7B died at approximately three months of age. Additionally, the copper content in the livers of rabbits at the onset of WD increased nine-fold, a level similar to the five-fold increase observed in humans with WD. Thus, the efficiency of precision point mutations increases when RNAs are injected into zygotes at earlier stages, and the ATP7B mutant rabbits are a potential model for human WD disease with applications in pathological analysis, clinical treatment and gene therapy research.
Highlights
Diseases are induced by enhanced or varied gene functions derived from SNPs26,27
The dysfunction of ATP7B contributes to Wilson disease (WD), an autosomal recessive genetic disorder of copper metabolism caused by a mutation in the ATP7B gene[34,35,36]
This research was based on the use of micro-manipulation technology combined with an efficient and specific CRISPR/Cas[9] system to make the ATP7B gene point mutation occur at the high homology site of the 780th amino acid arginine through homologous recombination
Summary
Diseases are induced by enhanced or varied gene functions derived from SNPs26,27. These hereditary diseases cannot be simulated by KO animals. Several transgenic or low-density lipoprotein receptor gene mutation rabbit models have been used for the study of lipoproteins and atherosclerosis because the lipid metabolism of rabbits is similar to that of humans[27,28,29]. The defined point mutations in the rabbit ATP7B gene were derived by microinjecting synthesized RNAs with ssODN donor sequences into zygotes, and the efficiency of homology-directed knock-in of point mutations was enhanced when RNAs were injected into younger zygotes
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