Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
Cell. 2013 Sep 12;154(6):1370-9. doi: 10.1016/j.cell.2013.08.022. Epub 2013 Aug 29.
The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.
II 型细菌 CRISPR/Cas 系统是一种新型的基因组工程技术，具有多重基因靶向的优势。在这里，我们通过将 Cas9 mRNA 和不同的向导 RNA（sgRNA）与不同大小的 DNA 载体一起注射到受精卵中，创建了报告基因和条件性突变体小鼠。通过这种一步法，我们生成了携带 Nanog、Sox2 和 Oct4 基因标签或荧光报告构建体以及 Mecp2 条件性突变体小鼠的小鼠。此外，我们使用靶向 Mecp2 基因两个不同位点的 sgRNA，产生了携带约 700bp 预测缺失的小鼠。最后，我们分析了基因修饰小鼠和 ESC 系中五个 sgRNA 的潜在脱靶，仅在少数情况下发现了脱靶突变。
