Efficient production and transmission of CRISPR/Cas9-mediated mutant alleles at the IG-DMR via generation of mosaic mice using a modified 2CC method
Efficient production and transmission of CRISPR/Cas9-mediated mutant alleles at the IG-DMR via generation of mosaic mice using a modified 2CC method
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DOI：
10.1038/s41598-019-56676-5
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发表时间：
2019-12-27
影响因子：
4.6
通讯作者：
Takada, Shuji
中科院分区：
文献类型：
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作者：
Hara, Satoshi;Terao, Miho;Takada, Shuji
利用基因组编辑技术构建突变型印记控制区(ICR)小鼠是阐明ICR功能的重要途径。IG-DMR是Dlk1-dio3印迹结构域中的ICR，它包含两个亲本等位基因中对胚胎发育至关重要的功能区。这种方法的一个缺点是，如果IG-DMR在父亲或母亲的等位基因中发生突变，印记基因的异常表达可能会导致胚胎死亡。为了克服这个问题，我们使用2CC方法产生了含有修饰的IG-DMR等位基因的细胞和野生型细胞的嵌合体小鼠，这种方法允许将CRISPR/Cas9结构显微注射到2-细胞胚胎的卵裂球中。与使用标准方案获得的幼崽相比，这种方法提高了创始幼崽的出生率。我们还成功地产生了嵌合体小鼠，其中IG-DMR中的串联重复序列已被同源定向修复所取代。此外，由于IG-DMR的低甲基化，被替换的等位基因的父系传递导致印记基因的异常表达，表明被替换的等位基因重现了我们的缺失模型。我们的结果表明，这种方法对于突变小鼠的产生是有用的，在突变小鼠中，正常发育所必需的基因组位置已经被遗传编辑。
Generation of mutant imprinting control region (ICR) mice using genome editing is an important approach for elucidating ICR functions. IG-DMR is an ICR in the Dlk1-Dio3 imprinted domain that contains functional regions-in both parental alleles-that are essential for embryonic development. One drawback of this approach is that embryonic lethality can occur from aberrant expression of the imprinted genes if IG-DMR gets mutated in either the paternal or maternal allele. To overcome this problem, we generated mosaic mice that contained cells with modified IG-DMR alleles and wild-type cells using the 2CC method that allowed for microinjection of the CRISPR/Cas9 constructs into a blastomere of 2-cell embryos. This method improved the birth rate of the founder pups relative to that obtained using the standard protocol. We also successfully produced mosaic mice in which the tandem repeat array sequence in the IG-DMR had been replaced by homology directed repair. Additionally, paternal transmission of the replaced allele caused aberrant expression of the imprinted genes due to hypomethylation of the IG-DMR, indicating that the replaced allele recapitulated our deletion model. Our results indicate that this method is useful for the generation of mutant mice in which a genomic locus essential for normal development has been genetically edited.