PRJNA731544
Analysis of NHEJ-based DNA repair after CRISPR-mediated DNA cleavage
DNA double-strand breaks (DSBs) are most lethal DNA damage which not only can cause several diseases such as cancer, but also can give rise to cell death. In eukaryotic cells, DSBs are repaired through homology-directed repair (HDR), non-homologous end joining (NHEJ), or microhomology-mediated end joining (MMEJ). Especially, NHEJ is error-prone pathway, so that genetic modification such as insertions and deletions (indels) can easily occur at DSB during NHEJ-mediated repair. Thanks to this property, the NHEJ pathway has been exploited for gene editing tools including the CRISPR-Cas9 system. However, the features of the NHEJ-mediated DSB repair have not been studied well. Here, we assess the accuracy of the NHEJ-mediated repair of DSB generated in the genome by the Cas9 nuclease in human-originated cells. To prevent subsequent DSB generation following a previous flawless DSB repair, which can make the accuracy underestimated, we added a modified oligomer which can be inserted into the DSB during the NHEJ-mediated repair. Through sequencing the oligomer and the target site after DSB repair, we found that the repair accuracy is sequence dependent and lower at the DSB end proximal to PAM. In addition, we identified that insertion mutation is correlated to the repair accuracy. These results suggest that the process of the NHEJ-mediated DSB repair is largely affected by the target sequence which may be a determinant of the mutation type.