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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 t...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8232687/ https://www.ncbi.nlm.nih.gov/pubmed/34203807 http://dx.doi.org/10.3390/ijms22126397 |
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author | Song, Beomjong Yang, Soyeon Hwang, Gue-Ho Yu, Jihyeon Bae, Sangsu |
author_facet | Song, Beomjong Yang, Soyeon Hwang, Gue-Ho Yu, Jihyeon Bae, Sangsu |
author_sort | Song, Beomjong |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8232687 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-82326872021-06-26 Analysis of NHEJ-Based DNA Repair after CRISPR-Mediated DNA Cleavage Song, Beomjong Yang, Soyeon Hwang, Gue-Ho Yu, Jihyeon Bae, Sangsu Int J Mol Sci Article 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. MDPI 2021-06-15 /pmc/articles/PMC8232687/ /pubmed/34203807 http://dx.doi.org/10.3390/ijms22126397 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Song, Beomjong Yang, Soyeon Hwang, Gue-Ho Yu, Jihyeon Bae, Sangsu Analysis of NHEJ-Based DNA Repair after CRISPR-Mediated DNA Cleavage |
title | Analysis of NHEJ-Based DNA Repair after CRISPR-Mediated DNA Cleavage |
title_full | Analysis of NHEJ-Based DNA Repair after CRISPR-Mediated DNA Cleavage |
title_fullStr | Analysis of NHEJ-Based DNA Repair after CRISPR-Mediated DNA Cleavage |
title_full_unstemmed | Analysis of NHEJ-Based DNA Repair after CRISPR-Mediated DNA Cleavage |
title_short | Analysis of NHEJ-Based DNA Repair after CRISPR-Mediated DNA Cleavage |
title_sort | analysis of nhej-based dna repair after crispr-mediated dna cleavage |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8232687/ https://www.ncbi.nlm.nih.gov/pubmed/34203807 http://dx.doi.org/10.3390/ijms22126397 |
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