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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Elevated expression of exogenous RAD51 enhances the CRISPR/Cas9-mediated genome editing efficiency

  • Seo Jung Park (Department of Life Science, Chung-Ang University) ;
  • Seobin Yoon (Department of Life Science, Chung-Ang University) ;
  • Eui-Hwan Choi (Department of Life Science, Chung-Ang University) ;
  • Hana Hyeon (Department of Life Science, Chung-Ang University) ;
  • Kangseok Lee (Department of Life Science, Chung-Ang University) ;
  • Keun Pil Kim (Department of Life Science, Chung-Ang University)
  • Received : 2022.09.21
  • Accepted : 2022.12.11
  • Published : 2023.02.28
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Abstract

Genome editing using CRISPR-associated technology is widely used to modify the genomes rapidly and efficiently on specific DNA double-strand breaks (DSBs) induced by Cas9 endonuclease. However, despite swift advance in Cas9 engineering, structural basis of Cas9-recognition and cleavage complex remains unclear. Proper assembly of this complex correlates to effective Cas9 activity, leading to high efficacy of genome editing events. Here, we develop a CRISPR/Cas9-RAD51 plasmid constitutively expressing RAD51, which can bind to single-stranded DNA for DSB repair. We show that the efficiency of CRISPR-mediated genome editing can be significantly improved by expressing RAD51, responsible for DSB repair via homologous recombination (HR), in both gene knock-out and knock-in processes. In cells with CRISPR/Cas9-RAD51 plasmid, expression of the target genes (cohesin SMC3 and GAPDH) was reduced by more than 1.9-fold compared to the CRISPR/Cas9 plasmid for knock-out of genes. Furthermore, CRISPR/Cas9-RAD51 enhanced the knock-in efficiency of DsRed donor DNA. Thus, the CRISPR/Cas9-RAD51 system is useful for applications requiring precise and efficient genome edits not accessible to HR-deficient cell genome editing and for developing CRISPR/Cas9-mediated knockout technology.

Keywords

Acknowledgement

This research was supported by the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning (2020R1A2C2011887; 2018R1A5A1025077) and the Chung-Ang University Graduate Research Scholarship in 2021.

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