CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) mutations as gene knockouts at targeted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20–33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as EGFP, mCherry, Cre, and genes of interest, at the other exon site. These findings indicate that this combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats.

CRISPR-Cas9被广泛用于小鼠和大鼠的基因靶向。在受精卵中占主导地位的非同源末端连接（NHEJ）修复途径有效地诱导插入或缺失（indel）突变，作为基因敲除的靶位，而基因敲入（KIs）通过同源性定向修复（HDR） ）很难生成。在这项研究中，我们使用了带有Cas9和两个单向导RNA的双链DNA（dsDNA）供体模板，一个设计用于切割目标基因组序列，另一个设计用于切割侧翼基因组区域和dsDNA质粒的一个同源臂。 ，导致G0幼犬的KI效率达到20-33％。G0 KI小鼠在一个设计为内含子区域的靶向位点携带NHEJ依赖的indel突变，以及各种供体盒的HDR依赖的精确KI，其跨度为1-5 kbp，例如EGFP，mCherry，Cre和感兴趣的基因，在另一个外显子位点。这些发现表明，这种由CRISPR-Cas9系统介导的NHEJ和HDR的组合方法促进了小鼠和大鼠中质粒DNA盒的有效和精确的KIs。