Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering.
Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering.
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DOI：
10.1038/srep04513
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发表时间：
2014-03-28
影响因子：
4.6
通讯作者：
Hatada I
中科院分区：
文献类型：
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作者：
Horii T;Arai Y;Yamazaki M;Morita S;Kimura M;Itoh M;Abe Y;Hatada I
CRISPR/Cas系统，其中Cas9内切核酸酶和与靶标互补的指导RNA足以用于靶DNA的RNA引导切割，是最近开发的用于在各种动物模型中靶向基因破坏的强大新方法。然而，很少有验证的显微注射方法产生敲除小鼠使用这种方法。在这里，我们报告了CRISPR/Cas系统的显微注射方法的验证。我们比较了三种注射方法：（1）将DNA注射到原核中，（2）将RNA注射到原核中，和（3）将RNA注射到细胞质中。我们发现，注射RNA到细胞质中是最有效的方法，在数量的可行的囊胚期胚胎和足月的幼崽产生。该方法还显示出最佳的总体敲除效率。
The CRISPR/Cas system, in which the Cas9 endonuclease and a guide RNA complementary to the target are sufficient for RNA-guided cleavage of the target DNA, is a powerful new approach recently developed for targeted gene disruption in various animal models. However, there is little verification of microinjection methods for generating knockout mice using this approach. Here, we report the verification of microinjection methods of the CRISPR/Cas system. We compared three methods for injection: (1) injection of DNA into the pronucleus, (2) injection of RNA into the pronucleus, and (3) injection of RNA into the cytoplasm. We found that injection of RNA into the cytoplasm was the most efficient method in terms of the numbers of viable blastocyst stage embryos and full-term pups generated. This method also showed the best overall knockout efficiency.