Department of Laboratory Animal Science, School of Medicine, Shanghai Jiao Tong University, 200025, Shanghai, China.
School of Life Science and Technology, Shanghai Tech University, 201210, Shanghai, China.
Sci Rep. 2018 Jan 22;8(1):1332. doi: 10.1038/s41598-018-19774-4.
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.
CRISPR/Cas9 最近被开发为一种高效的基因组工程工具。兔子是代谢性疾病研究的合适动物模型。在这项研究中，我们通过使用 CRISPR/Cas9 系统结合单链 DNA 寡核苷酸（ssODNs），生成了 ATP7B 定点突变兔，以模拟威尔逊病（WD）中亚洲人主要的突变类型（p.Arg778Leu）。当受精卵在 hCG 处理后 14 小时注射时，精确点突变的效率为 52.94%，明显高于 hCG 处理后 19 小时注射的受精卵（14.29%）。携带突变 ATP7B 等位基因的兔子在大约三个月大时死亡。此外，WD 发病时兔子肝脏中的铜含量增加了九倍，与 WD 患者观察到的五倍增加相似。因此，当 RNA 更早地注入受精卵时，精确点突变的效率会增加，而 ATP7B 突变兔是一种潜在的人类 WD 疾病模型，可应用于病理分析、临床治疗和基因治疗研究。
