Quantitative assessment of HR and NHEJ activities via CRISPR/Cas9-induced oligodeoxynucleotide-mediated DSB repair.

ABSTRACT

Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two major mechanisms for the repair of DNA double-strand breaks (DSBs) in eukaryotic cells. Previously, we designed an assay for detecting NHEJ activity by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, however, this approach cannot be used to predict the activity of HR repair. Hence, we developed a novel method that is capable of quantitatively measuring both HR and NHEJ activities via CRISPR/Cas9-induced oligodeoxynucleotide (ODN)-mediated DSB repair. In the present experimental procedures, the CRISPR/Cas9 plasmid was cotransfected with single-stranded ODN (ssODN) or blunt-ended double-stranded ODN (dsODN), both of which harbored a unique marker sequence. After the induction of site-specific DSBs by CRISPR/Cas9 system, the ssODN, functioned as the donor template for HR repair, could insert the marker sequence into the DSB sites, while the dsODN was embedded in the DSB sites through NHEJ pathway. Next, by means of PCR analysis using a specific primer for the marker sequence and the primers that flank the DSB sites, the relative amount of integrated marker sequence in the genomic DNA could be quantitatively determined. The correlation between the marker sequence abundance and the HR and NHEJ activities was confirmed by using the selective HR and NHEJ inhibitors. This accessible and rapid quantitative assay for HR and NHEJ activities might be useful for the future research of the DSB repair mechanisms.