Cas9融合RAD18因子提高HEK293T细胞的定点敲入效率
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国家转基因重大专项(2016ZX08006002);广东特支本土创新创业团队(2019BT02N630)


Increase of knock-in efficiency in HEK293T cells by fusing RAD18 factor to Cas9
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    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RAD51RAD18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 of RAD51/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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黄广燕,王豪强,李国玲,吴珍芳,张献伟. Cas9融合RAD18因子提高HEK293T细胞的定点敲入效率[J].华南农业大学学报,2021,42(5):8-18

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  • 收稿日期:2020-11-25
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  • 在线发布日期: 2021-08-02