Elevated expression of exogenous RAD51 enhances the CRISPR/Cas9-mediated genome editing efficiency

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 singlestranded 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.

Recent Advances in the Production of Genome-Edited Animals Using <i>i</i>-GONAD, a Novel <i>in vivo</i> Genome Editing System, and Its Possible Use for the Study of Female Reproductive Systems

“…Notably, the combined use of purified RAD51 protein (RAD51 recombinase) and CRISPR-Cas9 reagents has recently been shown to significantly increase the efficiency of homozygous KI in mouse embryos [65]. Park et al [66] reported similar observations. Notably, Ma et al [67] recently developed miCas9 by fusing a minimal motif consisting of 36 amino acids (encoded by BReast CAncer gene 1 exon 27) to Streptococcus pyogenes Cas9 (SpCas9).…”