Production of Wilson Disease Model Rabbits with Homology-Directed Precision Point Mutations in the ATP7B Gene Using the CRISPR/Cas9 System

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.

Clinical features and mutational analysis in 114 young children with Wilson disease from South China

“…Wu et al () also reported that p.R778L homozygous patients had early onset of hepatic damage. WD rabbit model homozygous for p.R778L died of severe hepatic damage at 3 months of age (Jiang et al, ). In vitro investigation revealed that the R778L mutation may disrupt the subcellular localization and trafficking of the ATP7B protein, reducing copper excretion, and resulting in copper accumulation (Zhu, Dong, Ni, & Wu, ).…”

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects

“…[205][206][207] Other studies have utilized endonucleases to generate different kinds of immunodeficient animal models that were previously unable to be established due to a lack of effective genetic modification. [207][208][209][210] As a result of engineered nucleasemediated editing of genomic modifications, other animal disease models have been developed, simulating Rett syndrome, 211 hereditary deafness, 212 Wilson disease, 213 Laron syndrome, 214 Niemann-Pick disease, 215 Netherton syndrome, 216 and so on. Advances in genome editing technologies will further expand the application of animal models in disease mechanism research and treatment development.…”

Using CRISPR/Cas9 to model human liver disease

“…58,59 Wilson disease Jiang et al created precision point mutations, using CRISPR technology in rabbits, to develop a model of Wilson disease (WD). 60 WD is an autosomal recessive genetic disorder caused by mutations in the membrane copper transporter gene, ATP7B. 61,62 The authors targeted the Atp7b gene with sgRNAs and provided a single-stranded donor oligonucleotide template for homology-directed repair, to obtain the missense mutation Arg778Leu, a common disease mutation in Asian populations.…”

“…61,62 The authors targeted the Atp7b gene with sgRNAs and provided a single-stranded donor oligonucleotide template for homology-directed repair, to obtain the missense mutation Arg778Leu, a common disease mutation in Asian populations. 60 When comparing zygotes obtained from donor rabbits 14 h or 19 h after human chorionic gonadotropin treatment, the authors found that injections performed in pronuclear embryos at earlier stages resulted in higher rates of point mutation (over 50%) and reduced gene knockout. Atp7b mutant rabbits exhibited copper accumulation in the liver and kidney, as observed in human WD, and died at an early age of 3 months.…”