Analysis of nhej-based dna repair after crispr-mediated dna cleavage

Beomjong Song, Soyeon Yang, Gue Ho Hwang, Jihyeon Yu, Sangsu Bae

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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.

Original languageEnglish
Article number6397
JournalInternational Journal of Molecular Sciences
Volume22
Issue number12
DOIs
StatePublished - 2021 Jun 2

Keywords

  • CRISPR
  • DNA double-strand break
  • Genome editing
  • NHEJ-based DNA repair
  • Non-homologous end joining
  • Repair accuracy

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