Institut Pasteur, CNRS, Unité de Génétique Fonctionnelle de la Souris, UMR 3738, Department of Developmental &Stem Cell Biology, 25 rue du docteur Roux, F-75015 Paris.
Sci Rep. 2017 Feb 17;7:42661. doi: 10.1038/srep42661.
Microinjection of the CRISPR/Cas9 system in zygotes is an efficient and comparatively fast method to generate genetically modified mice. So far, only few knock-in mice have been generated using this approach, and because no systematic study has been performed, parameters controlling the efficacy of CRISPR/Cas9-mediated targeted insertion are not fully established. Here, we evaluated the effect of several parameters on knock-in efficiency changing only one variable at a time. We found that knock-in efficiency was dependent on injected Cas9 mRNA and single-guide RNA concentrations and that cytoplasmic injection resulted in more genotypic complexity compared to pronuclear injection. Our results also indicated that injection into the pronucleus compared to the cytoplasm is preferable to generate knock-in alleles with an oligonucleotide or a circular plasmid. Finally, we showed that Cas9D10A nickase variant was less efficient than wild-type Cas9 for generating knock-in alleles and caused a higher rate of mosaicism. Thus, our study provides valuable information that will help to improve the future production of precise genetic modifications in mice.
将 CRISPR/Cas9 系统微注射到受精卵中是一种高效且相对快速的方法，可以用来生成基因修饰的小鼠。到目前为止，仅使用这种方法生成了少数几个基因敲入小鼠，而且由于没有进行系统的研究，因此控制 CRISPR/Cas9 介导的靶向插入效率的参数尚未完全建立。在这里，我们评估了几个参数对基因敲入效率的影响，每次只改变一个变量。我们发现，基因敲入效率取决于注射的 Cas9 mRNA 和单链向导 RNA 的浓度，并且细胞质注射比原核注射导致更多的基因型复杂性。我们的结果还表明，与细胞质注射相比，将 Cas9 注射到原核中更有利于使用寡核苷酸或环形质粒生成基因敲入等位基因。最后，我们表明 Cas9D10A 切口酶变体用于生成基因敲入等位基因的效率低于野生型 Cas9，并导致更高的嵌合率。因此，我们的研究提供了有价值的信息，将有助于提高未来在小鼠中进行精确遗传修饰的效率。
