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    Cas9融合RAD18因子提高HEK293T细胞的定点敲入效率

    Increase of knock-in efficiency in HEK293T cells by fusing RAD18 factor to Cas9

      摘要
    • 摘要:
      目的  在哺乳动物细胞中，RAD51和RAD18是调节同源定向修复和非同源末端连接的关键因子。本研究目的在于探讨这2个因子在eSpCas9系统中对HEK293T细胞的定点敲入(Knock-in, KI)效率的影响，进而提高KI效率。
      方法  通过构建eSpCas9-RAD51、eSpCas9-RAD18融合蛋白以及过表达RAD51、RAD18载体，比较它们的KI效率差异。
      结果  eSpCas9-RAD18系统可以显著提高HEK293T细胞KI效率，约为原来eSpCas9系统的1.4~1.9倍(P<0.01)，而eSpCas9-RAD51系统和过表达RAD51/RAD18对KI效率无显著提高作用。
      结论  本研究构建的eSpCas9-RAD18系统可以有效地提高HEK293T细胞的KI效率，可为基因编辑、基因治疗及定点转基因提供一种新的辅助整合工具。

       

      Abstract:
      Objective  In mammalian cells, RAD51 and RAD18 are the key factors for regulating the relationship between non-homologous end joining and homology-directed repair. The purpose of this study was to explore the effects of these two factors on the knock-in (KI) efficiency in HEK293T cell lines by eSpCas9 system, and improve the KI efficiency.
      Method  eSpCas9-RAD51, eSpCas9-RAD18 fusion proteins and RAD51, RAD18 overexpression vectors were constructed to compare their difference of KI efficiency.
      Result  Only the eSpCas9-RAD18 system could significantly increase the KI efficiency of HEK293T cells, which was about 1.4~1.9 times that of the original eSpCas9 system (P<0.01). The eSpCas9-RAD51 system and overexpression ofRAD51/RAD18 did not improve the KI efficiency.
      Conclusion  The eSpCas9-RAD18 system constructed in this study can effectively improve the KI efficiency in HEK293T cells, and provides a novel auxiliary integration tool for gene editing, gene therapy and site-specific transgenesis.

       

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