Analysis of NHEJ-Based DNA Repair after CRISPR-Mediated DNA Cleavage.

Genome editing using CRISPR-Cas9 nucleases is based on the repair of the 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, it is thought that the NHEJ pathway is dominant and occurs throughout a cell cycle. NHEJ-based DSB repair is known to be error-prone; however, there are few studies that delve into it deeply in endogenous genes. Here, we quantify the degree of NHEJ-based DSB repair accuracy (termed NHEJ accuracy) in human-originated cells by incorporating exogenous DNA oligonucleotides. Through an analysis of joined sequences between the exogenous DNA and the endogenous target after DSBs occur, we determined that the average value of NHEJ accuracy is approximately 75% in maximum in HEK 293T cells. In a deep analysis, we found that NHEJ accuracy is sequence-dependent and the value at the DSB end proximal to a protospacer adjacent motif (PAM) is relatively lower than that at the DSB end distal to the PAM. In addition, we observed a negative correlation between the insertion mutation ratio and the degree of NHEJ accuracy. Our findings would broaden the understanding of Cas9-mediated genome editing.

CRISPR-sub: Analysis of DNA substitution mutations caused by CRISPR-Cas9 in human cells.

CRISPR-Cas9 induces DNA cleavages at desired target sites in a guide RNA-dependent manner; DNA editing occurs through the resulting activity of DNA repair processes including non-homologous end joining (NHEJ), which is dominant in mammalian cells. NHEJ repair frequently causes small insertions and deletions (indels) near DNA cleavage sites but only rarely causes nucleotide substitutions. High-throughput sequencing is the primary means of assessing indel and substitution frequencies in bulk populations of cells in the gene editing field. However, it is difficult to detect bona fide substitutions, which are embedded among experimentally-induced substitution errors, in high-throughput sequencing data. Here, we developed a novel analysis method, named CRISPR-Sub, to statistically detect Cas9-mediated substitutions in high-throughput sequencing data by comparing Mock- and CRISPR-treated samples. We first pinpointed 'hotspot positions' in target sequences at which substitution mutations were quantitatively observed much more often (p > 0.001) in CRISPR- versus Mock-treated samples. We refer to the substitution mutations in defined hotspot positions as 'apparent substitutions' and ultimately calculated 'apparent substitution frequencies' for each target. By examining 51 endogenous target sites in HeLa cells, we found that the average apparent substitution frequency was 0.8% in all queries, that apparent substitutions frequently occur near CRISPR-Cas9 cleavage sites, and that nucleotide conversion showed no meaningful nucleotide preference patterns. Furthermore, we generated NHEJ-inhibited cell lines (LIG4-/- ) by knockout of the gene encoding ligase IV and found that the apparent substitution frequencies were significantly decreased in LIG4-/- cells, strongly suggesting that DNA substitutions are generated by the NHEJ pathway.