Exome sequencing and CRISPR/Cas genome editing identify mutations of ZAK as a cause of limb defects in humans and mice.
Exome sequencing and CRISPR/Cas genome editing identify mutations of ZAK as a cause of limb defects in humans and mice.
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DOI：
10.1101/gr.199430.115
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发表时间：
2016-02
期刊：
影响因子：
7
通讯作者：
Borck G
中科院分区：
文献类型：
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作者：
Spielmann M;Kakar N;Tayebi N;Leettola C;Nürnberg G;Sowada N;Lupiáñez DG;Harabula I;Flöttmann R;Horn D;Chan WL;Wittler L;Yilmaz R;Altmüller J;Thiele H;van Bokhoven H;Schwartz CE;Nürnberg P;Bowie JU;Ahmad J;Kubisch C;Mundlos S;Borck G
CRISPR/Cas技术能够实现靶向基因组编辑和快速生成转基因动物模型，用于研究人类遗传疾病。在这里，我们描述了一种常染色体隐性遗传的人类疾病，在两个不相关的家庭，其特征是一个分裂的脚缺陷，指甲异常的手，听力损失，由于突变破坏SAM结构域的蛋白激酶ZAK。ZAK是MAPKKK家族的成员，在肢体发育中没有已知的作用。我们发现Zak在发育中的肢体中表达，并且CRISPR/Cas介导的两种Zak亚型的敲除在小鼠中具有胚胎致死性。相反，SAM结构域的缺失诱导与Trp 63（一种已知的裂手/裂足畸形疾病基因）下调相关的复杂后肢缺陷。我们的研究结果将ZAK确定为哺乳动物肢体模式的关键参与者，并证明了CRISPR/Cas基因组编辑的快速实用性，可在<10周内将因果关系分配给小鼠中的人类突变。
The CRISPR/Cas technology enables targeted genome editing and the rapid generation of transgenic animal models for the study of human genetic disorders. Here we describe an autosomal recessive human disease in two unrelated families characterized by a split-foot defect, nail abnormalities of the hands, and hearing loss, due to mutations disrupting the SAM domain of the protein kinase ZAK. ZAK is a member of the MAPKKK family with no known role in limb development. We show that Zak is expressed in the developing limbs and that a CRISPR/Cas-mediated knockout of the two Zak isoforms is embryonically lethal in mice. In contrast, a deletion of the SAM domain induces a complex hindlimb defect associated with down-regulation of Trp63, a known split-hand/split-foot malformation disease gene. Our results identify ZAK as a key player in mammalian limb patterning and demonstrate the rapid utility of CRISPR/Cas genome editing to assign causality to human mutations in the mouse in <10 wk.