Mashiko Daisuke, Fujihara Yoshitaka, Satouh Yuhkoh, Miyata Haruhiko, Isotani Ayako, Ikawa Masahito
1] Research Institute for Microbial Diseases [2] Graduate School of Medicine [3].
Sci Rep. 2013 Nov 27;3:3355. doi: 10.1038/srep03355.
CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 (hCas) mRNA and single guide RNA into fertilized eggs. Here we inject the circular plasmids expressing hCas9 and sgRNA into mouse zygotes and obtained mutant mice within a month. When we targeted the Cetn1 locus, 58.8% (10/17) of the pups carried the mutations and six of them were homozygously mutated. Co-injection of the plasmids targeting different loci resulted in the successful removal of the flanked region in two out of three mutant pups. The efficient mutagenesis was also observed at the Prm1 locus. Among the 46 offspring carrying CRISPR/Cas plasmid mediated mutations, only two of them carried the hCas9 transgene. The pronuclear injection of circular plasmid expressing hCas9/sgRNA complex is a rapid, simple, and reproducible method for targeted mutagenesis.
CRISPR/Cas介导的基因组编辑已在哺乳动物细胞中成功得到证实，并且通过将人源化Cas9（hCas）mRNA和单向导RNA注射到受精卵中，报道了其在生成突变小鼠方面的进一步应用。在此，我们将表达hCas9和sgRNA的环状质粒注射到小鼠受精卵中，并在一个月内获得了突变小鼠。当我们靶向Cetn1基因座时，58.8%（10/17）的幼崽携带突变，其中六只为纯合突变。共注射靶向不同基因座的质粒导致三只突变幼崽中有两只成功切除了侧翼区域。在Prm1基因座也观察到了高效的诱变作用。在携带CRISPR/Cas质粒介导突变的46只后代中，只有两只携带hCas9转基因。注射表达hCas9/sgRNA复合物的环状质粒到原核是一种快速、简单且可重复的靶向诱变方法。
