genOway, Lyon, 69007, France.
Sci Rep. 2019 Mar 5;9(1):3486. doi: 10.1038/s41598-019-40181-w.
The CRISPR/Cas9 gene editing tool enables accessible and efficient modifications which (re)ignited molecular research in certain species. However, targeted integration of large DNA fragments using CRISPR/Cas9 can still be challenging in numerous models. To systematically compare CRISPR/Cas9's efficiency to classical homologous recombination (cHR) for insertion of large DNA fragments, we thoroughly performed and analyzed 221 experiments targeting 128 loci in mouse ES cells. Although both technologies proved efficient, CRISPR/Cas9 yielded significantly more positive clones as detected by overlapping PCRs. It also induced unexpected rearrangements around the targeted site, ultimately rendering CRISPR/Cas9 less efficient than cHR for the production of fully validated clones. These data show that CRISPR/Cas9-mediated recombination can induce complex long-range modifications at targeted loci, thus emphasizing the need for thorough characterization of any genetically modified material obtained through CRISPR-mediated gene editing before further functional studies or therapeutic use.
CRISPR/Cas9 基因编辑工具可实现便捷高效的修饰，这重新激发了某些物种的分子研究。然而，在众多模型中，使用 CRISPR/Cas9 实现大片段 DNA 的靶向整合仍然具有挑战性。为了系统地比较 CRISPR/Cas9 与经典同源重组（cHR）在大片段 DNA 插入方面的效率，我们在小鼠胚胎干细胞（ES 细胞）的 128 个靶位点上彻底进行并分析了 221 项实验。虽然两种技术都被证明是有效的，但重叠 PCR 检测显示，CRISPR/Cas9 产生的阳性克隆明显更多。它还会在靶位点周围诱导意想不到的重排，最终导致 CRISPR/Cas9 的效率低于 cHR，无法产生完全有效的克隆。这些数据表明，CRISPR/Cas9 介导的重组可在靶位点诱导复杂的长距离修饰，因此在进一步进行功能研究或治疗用途之前，需要对通过 CRISPR 介导的基因编辑获得的任何遗传修饰材料进行彻底的特征描述。
