Analysis of NHEJ-based DNA repair after CRISPR-mediated DNA cleavage. Homo sapiens

Genome editing using CRISPR-Cas9 nucleases is based on the repair of DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ), or microhomology-mediated end joining (MMEJ) pathways. Among these, the NHEJ pathway has been thought to be dominant and occurred throughout a cell cycle. It is known that the NHEJ-based DSB repair is error-prone, but it has not been measured how accurate it is, especially in endogenous genome. Here, we quantify the degree of NHEJ-based DSB repair accuracy (NHEJ accuracy) in human-originated cells by incorporating exogenous DNA oligonucleotides. Through analyzing joined sequences between the exogenous DNA and endogenous target after DSBs occur, we obtained that the average value of the NHEJ accuracy is approximately 80%. In a deep analysis, we found that the NHEJ accuracy is sequence-dependent and the value at DSB end proximal to a protospacer adjacent motif (PAM) is relatively lower than that at DSB end distal to PAM. In addition, we observed that insertion mutation ratio is correlated to the NHEJ accuracy. Our findings can broaden the understanding of Cas9-mediated genome editing.