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

HUANG Guangyan; WANG Haoqiang; LI Guoling; WU Zhenfang; ZHANG Xianwei

doi:10.7671/j.issn.1001-411X.202011028

Journal of South China Agricultural University > 2021 > 42(5): 8-18. > DOI: 10.7671/j.issn.1001-411X.202011028

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

1.
College of Animal Science, South China Agricultural University/National Engineering Research Center for Breeding Swine Industry, Guangzhou 510642, China
2.
Wens Foodstuff Co., Ltd., Yunfu 527400, China

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Received Date: November 24, 2020
Available Online: May 17, 2023

Graphical Abstract

Abstract

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.
- gene editing,
- CRISPR/Cas9 system,
- gene knock-in,
- RAD18,
- fusion protein

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References (35)

References

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

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