Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9
Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9
复制标题
使用 CRISPR/Cas9 对恒河猴的肌营养不良蛋白基因进行功能破坏。
DOI：
10.1093/hmg/ddv120
复制
发表时间：
2015-07-01
影响因子：
3.5
通讯作者：
Li, Xiao-Jiang
中科院分区：
文献类型：
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作者：
Chen, Yongchang;Zheng, Yinghui;Li, Xiao-Jiang
CRISPR/Cas9 已被用于对包括非人类灵长类动物在内的多种物种的基因组进行基因改造。不幸的是，这项新技术确实会导致嵌合突变，而且我们还不知道这种突变是否会在功能上破坏目标基因或导致人类疾病中出现的病理。如果我们要使用 CRISPR/Cas9 生成人类疾病的大型动物模型，就必须解决这些问题。在这里，我们使用 CRISPR/Cas9 靶向猴肌营养不良蛋白基因，产生导致杜氏肌营养不良症 (DMD) 的突变，这是一种隐性 X 连锁形式的肌营养不良症。对相对靶向率的检查表明，Crispr/Cas9 靶向可能导致猴肌肉中高达 87% 的肌营养不良蛋白等位基因发生嵌合突变。此外，CRISPR/Cas9 会在雄性和雌性猴子中诱导突变，导致早期 DMD 中肌营养不良蛋白明显减少和肌肉退化。我们的研究结果表明，无论遗传模式如何，CRISPR/Cas9 都可以有效地生成人类疾病的猴子模型。新生的 Cas9 靶向猴子中存在退化的肌肉细胞表明，疾病早期阶段的治疗干预可能可以有效缓解肌病。
CRISPR/Cas9 has been used to genetically modify genomes in a variety of species, including non-human primates. Unfortunately, this new technology does cause mosaic mutations, and we do not yet know whether such mutations can functionally disrupt the targeted gene or cause the pathology seen in human disease. Addressing these issues is necessary if we are to generate large animal models of human diseases using CRISPR/Cas9. Here we used CRISPR/Cas9 to target the monkey dystrophin gene to create mutations that lead to Duchenne muscular dystrophy (DMD), a recessive X-linked form of muscular dystrophy. Examination of the relative targeting rate revealed that Crispr/Cas9 targeting could lead to mosaic mutations in up to 87% of the dystrophin alleles in monkey muscle. Moreover, CRISPR/Cas9 induced mutations in both male and female monkeys, with the markedly depleted dystrophin and muscle degeneration seen in early DMD. Our findings indicate that CRISPR/Cas9 can efficiently generate monkey models of human diseases, regardless of inheritance patterns. The presence of degenerated muscle cells in newborn Cas9-targeted monkeys suggests that therapeutic interventions at the early disease stage may be effective at alleviating the myopathy.