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Elevated expression of exogenous RAD51 enhances the CRISPR/Cas9-mediated genome editing efficiency
Seo Jung Park1 (Graduate Student), Seo-Bin Yoon1 (Graduate Student), Eui-Hwan Choi1 (Research worker), Hana Hyeon1 (Graduate Student), Kangseok Lee1 (Professor), Keun Pil Kim1,* (Professor)
1Life Science, Chung-Ang University
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. To overcome these limitations, 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.
Abstract, Accepted Manuscript(in press) [Submitted on September 21, 2022, Accepted on December 11, 2022]
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