In genome editing with CRISPR-Cas9, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homology-dependent repair (HDR). CtIP, a key protein in early steps of homologous recombination, is fused to Cas9 and stimulates transgene integration by HDR at the human AAVS1 safe harbor locus. A minimal N-terminal fragment of CtIP, designated HE for HDR enhancer, is sufficient to stimulate HDR and this depends on CDK phosphorylation sites and the multimerization domain essential for CtIP activity in homologous recombination. HDR stimulation by Cas9-HE, however, depends on the guide RNA used, a limitation that may be overcome by testing multiple guides to the locus of interest. The Cas9-HE fusion is simple to use and allows obtaining twofold or more efficient transgene integration than that with Cas9 in several experimental systems, including human cell lines, iPS cells, and rat zygotes.

中文翻译：

---

CtIP与Cas9的融合通过同源性依赖性修复增强了转基因整合。

在使用CRISPR-Cas9进行基因组编辑时，转基因整合通常仍然具有挑战性。在这里，我们提出了一种通过同源依赖修复（HDR）提高转基因整合效率的方法。CtIP是同源重组早期的关键蛋白，与Cas9融合，并通过人AAVS1安全港所在地的HDR刺激转基因整合。CtIP的最小N末端片段（称为HE代表HDR增强剂）足以刺激HDR，这取决于CDK磷酸化位点和同源重组中CtIP活性必不可少的多聚结构域。但是，Cas9-HE对HDR的刺激取决于所用的指导RNA，可以通过测试目标基因座的多个指导来克服这一局限性。