Precise and rapid validation of candidate gene by allele specific knockout with CRISPR/Cas9 in wild mice
Precise and rapid validation of candidate gene by allele specific knockout with CRISPR/Cas9 in wild mice
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通过 CRISPR/Cas9 在野生小鼠中进行等位基因特异性敲除，精确快速地验证候选基因
DOI：
10.3389/fgene.2019.00124
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发表时间：
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影响因子：
3.7
通讯作者：
Liaoxun Lu
中科院分区：
文献类型：
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作者：
Tianzhu Chao;Zhuangzhuang Liu;Yu Zhang;Lichen Zhang;Rong Huang;Le He;Yanrong Gu;Zhijun Chen;Qianqian Zheng;Lijin Shi;Wenping Zheng;Xinhui Qi;Eryan Kong;Zhongjian Zhang;Toby Lawrence;Yinming Liang;Liaoxun Lu
确定小鼠近交系间表型差异的致病基因是一个诱人的目标，这是了解哺乳动物病理生理的巨大遗传资源库。特别是，野生小鼠品系在数十万年的进化分化过程中获得了巨大的遗传变异。然而，在CRISPR/Cas9基因组编辑工具出现之前，验证非经典菌株的遗传变异非常困难。在这项研究中，我们首先描述了野生来源的PWD/PhJ亲本小鼠和F1杂交小鼠的T细胞表型，从一个杂交到C57BL/6 （B6）小鼠，我们分离了Chr2上的一个遗传位点，使用连锁定位和染色体替换小鼠。重要的是，我们验证了控制这种T细胞表型的功能基因Cd44的鉴定，通过等位基因特异性敲除PWD拷贝，使B6拷贝完全完整。我们使用具有显性表型的F1小鼠进行实验，通过设计仅针对PWD基因组DNA的sgRNA PAM序列，可以快速验证候选基因。我们获得了10只来自B6卵子的动物，这些动物与PWD精子细胞受精，并进行了CRISPR/Cas9基因靶向机制的显微注射。在F1杂交的新生儿中，80% （n=10）的小鼠出现了PWD来源的候选基因Cd44的等位基因特异性敲除，没有小鼠出现B6拷贝的错靶。在等位基因特异性敲除F1小鼠中，我们观察到T细胞表型完全恢复。因此，我们的研究提供了一种精确和快速的方法来功能验证基因，可以促进经典小鼠遗传学中的基因发现。更重要的是，由于我们成功地对小鼠进行了基因操作，等位基因特异性敲除可以使疾病等位基因失活，同时保持人类细胞中正常等位基因的完整。
It is a tempting goal to identify causative genes underlying phenotypic differences among inbred strains of mice, which is a huge reservoir of genetic resources to understand mammalian pathophysiology. In particular, the wild-derived mouse strains harbor enormous genetic variations that have been acquired during evolutionary divergence over hundreds of thousands of years. However, validating the genetic variation in non-classical strains was extremely difficult, until the advent of CRISPR/Cas9 genome editing tools. In this study, we first describe a T cell phenotype in both wild-derived PWD/PhJ parental mice and F1 hybrids, from a cross to C57BL/6 (B6) mice, and we isolate a genetic locus on Chr2, using linkage mapping and chromosome substitution mice. Importantly, we validate the identification of the functional gene controlling this T cell phenotype, Cd44, by allele specific knockout of the PWD copy, leaving the B6 copy completely intact. Our experiments using F1 mice with a dominant phenotype, allowed rapid validation of candidate genes by designing sgRNA PAM sequences that only target the DNA of the PWD genome. We obtained 10 animals derived from B6 eggs fertilized with PWD sperm cells which were subjected to microinjection of CRISPR/Cas9 gene targeting machinery. In the newborns of F1 hybrids, 80% (n=10) had allele specific knockout of the candidate gene Cd44 of PWD origin, and no mice showed mistargeting of the B6 copy. In the resultant allele-specific knockout F1 mice, we observe full recovery of T cell phenotype. Therefore, our study provided a precise and rapid approach to functionally validate genes that could facilitate gene discovery in classic mouse genetics. More importantly, as we succeeded in genetic manipulation of mice, allele specific knockout could provide the possibility to inactivate disease alleles while keeping the normal allele of the gene intact in human cells.