Elevated expression of exogenous RAD51 enhances the CRISPR/Cas9-mediated genome editing efficiency
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Genome editing using CRISPR-associated technology is widelyused to modify the genomes rapidly and efficiently on specificDNA double-strand breaks (DSBs) induced by Cas9 endonuclease. However, despite swift advance in Cas9 engineering,structural basis of Cas9-recognition and cleavage complex remainsunclear. Proper assembly of this complex correlates toeffective Cas9 activity, leading to high efficacy of genomeediting events. Here, we develop a CRISPR/Cas9-RAD51 plasmidconstitutively expressing RAD51, which can bind to singlestrandedDNA for DSB repair. We show that the efficiency ofCRISPR-mediated genome editing can be significantly improvedby expressing RAD51, responsible for DSB repair via homologousrecombination (HR), in both gene knock-out and knock-inprocesses. In cells with CRISPR/Cas9-RAD51 plasmid, expressionof the target genes (cohesin SMC3 and GAPDH) was reducedby more than 1.9-fold compared to the CRISPR/Cas9 plasmidfor knock-out of genes. Furthermore, CRISPR/Cas9-RAD51 enhancedthe knock-in efficiency of DsRed donor DNA. Thus, theCRISPR/Cas9-RAD51 system is useful for applications requiringprecise and efficient genome edits not accessible to HR-deficientcell genome editing and for developing CRISPR/Cas9-mediatedknockout technology.
Abstract
Genome editing using CRISPR-associated technology is widelyused to modify the genomes rapidly and efficiently on specificDNA double-strand breaks (DSBs) induced by Cas9 endonuclease. However, despite swift advance in Cas9 engineering,structural basis of Cas9-recognition and cleavage complex remainsunclear. Proper assembly of this complex correlates toeffective Cas9 activity, leading to high efficacy of genomeediting events. Here, we develop a CRISPR/Cas9-RAD51 plasmidconstitutively expressing RAD51, which can bind to singlestrandedDNA for DSB repair. We show that the efficiency ofCRISPR-mediated genome editing can be significantly improvedby expressing RAD51, responsible for DSB repair via homologousrecombination (HR), in both gene knock-out and knock-inprocesses. In cells with CRISPR/Cas9-RAD51 plasmid, expressionof the target genes (cohesin SMC3 and GAPDH) was reducedby more than 1.9-fold compared to the CRISPR/Cas9 plasmidfor knock-out of genes. Furthermore, CRISPR/Cas9-RAD51 enhancedthe knock-in efficiency of DsRed donor DNA. Thus, theCRISPR/Cas9-RAD51 system is useful for applications requiringprecise and efficient genome edits not accessible to HR-deficientcell genome editing and for developing CRISPR/Cas9-mediatedknockout technology.
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