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    HUANG Guangyan, WANG Haoqiang, LI Guoling, et al. Increase of knock-in efficiency in HEK293T cells by fusing RAD18 factor to Cas9[J]. Journal of South China Agricultural University, 2021, 42(5): 8-18. DOI: 10.7671/j.issn.1001-411X.202011028
    Citation: HUANG Guangyan, WANG Haoqiang, LI Guoling, et al. Increase of knock-in efficiency in HEK293T cells by fusing RAD18 factor to Cas9[J]. Journal of South China Agricultural University, 2021, 42(5): 8-18. DOI: 10.7671/j.issn.1001-411X.202011028
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    Increase of knock-in efficiency in HEK293T cells by fusing RAD18 factor to Cas9

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      • 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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