Abstract
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.
Highlights
We measured and compared the accuracy of the repair by the non-homologous end joining (NHEJ) pathway on the doublestrand break (DSB) generated by the Cas9 nucleases in human-originated cells
Accuracy was 75% in maximum), (ii) the NHEJ-mediated DSB repair was typically more error-prone at the protospacer adjacent motif (PAM)-proximal end of the DSB and (iii) the fraction of insertions in the total mutations showed a negative correlation with the degree of NHEJ accuracy
Our results suggested that two ends of the DSB site might go through differently via clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 nucleases
Summary
The field of genome editing has grown rapidly as a result of the harnessing of programmable nucleases such as zinc-finger nucleases (ZFNs) [1], transcription activator-like effector nucleases (TALENs) [2], and clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) endonucleases [3,4,5]. The CRISPR-Cas nuclease has superseded others because it can target genes more in an RNA-guided way [3,4,5,6]. The programmable nucleases commonly generate double-strand breaks (DSBs) on the target DNA, resulting in genome editing through a cell’s repair system involving a homology-directed repair (HDR), a non-homologous end joining (NHEJ) and the Ku protein-independent non-canonical NHEJ pathway such as microhomology-mediated end joining (MMEJ) [7,8,9,10,11]. In contrast with the HDR pathway that is highly faithful and error-free, the NHEJ pathway is error-prone, and small nucleotide insertions and deletions (indels) occur frequently at the cleavage site [12]
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