Generation of an Oocyte-Specific Cas9 Transgenic Mouse for Genome Editing.
Generation of an Oocyte-Specific Cas9 Transgenic Mouse for Genome Editing.
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生成用于基因组编辑的卵母细胞特异性 Cas9 转基因小鼠。
DOI：
10.1371/journal.pone.0154364
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Lou X
中科院分区：
文献类型：
--
作者：
Zhang L;Zhou J;Han J;Hu B;Hou N;Shi Y;Huang X;Lou X
CRISPR/Cas9系统已经被开发为通过受精卵显微注射Cas9和sgRNA来工程化真核基因组的易于操作和可复用的方法，而制备用于显微注射的Cas9是费力的并且在实验中引入不一致性。在这里，我们描述了一种通过使用卵母细胞特异性Cas9转基因小鼠进行基因靶向的改进策略。通过这种小鼠品系，我们成功地通过将sgRNA注射到单细胞阶段的胚胎中来实现精确的基因靶向。通过综合分析，我们还显示该策略诱导的等位基因复杂性和脱靶突变明显低于Cas9 mRNA/sgRNA注射。因此，将sgRNA注射到卵母细胞特异性Cas9转基因小鼠胚胎中为小鼠基因组编辑提供了方便、有效和可靠的方法。
The CRISPR/Cas9 system has been developed as an easy-handle and multiplexable approach for engineering eukaryotic genomes by zygote microinjection of Cas9 and sgRNA, while preparing Cas9 for microinjection is laborious and introducing inconsistency into the experiment. Here, we describe a modified strategy for gene targeting through using oocyte-specific Cas9 transgenic mouse. With this mouse line, we successfully achieve precise gene targeting by injection of sgRNAs only into one-cell-stage embryos. Through comprehensive analysis, we also show allele complexity and off-target mutagenesis induced by this strategy is obviously lower than Cas9 mRNA/sgRNA injection. Thus, injection of sgRNAs into oocyte-specific Cas9 transgenic mouse embryo provides a convenient, efficient and reliable approach for mouse genome editing.