|
KMID : 0624620230560020102
|
|
BMB Reports
2023 Volume.56 No. 2 p.102 ~ p.107
|
|
|
Elevated expression of exogenous RAD51 enhances the CRISPR/Cas9-mediated genome editing efficiency
|
|
Park Seo-Jung
Yoon Seo-Bin
Choi Eui-Hwan
Hyeon Ha-Na
Lee Kang-Seok
Kim Keun-Pil
|
|
|
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.
|
|
|
KEYWORD
|
|
|
CRISPR-Cas9, DSB, Genome editing, Homologous recombination, RAD51
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
  
|
|