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Comprehensive Analysis of CRISPR/Cas9-Mediated Mutagenesis in Arabidopsis thaliana by Genome-Wide Sequencing

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been widely applied in functional genomics research and plant breeding. In contrast to the off-target studies of mammalian cells, there is little evidence for the common occurrence of of...

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Autores principales: Xu, Wenjie, Fu, Wei, Zhu, Pengyu, Li, Zhihong, Wang, Chenguang, Wang, Chaonan, Zhang, Yongjiang, Zhu, Shuifang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747142/
https://www.ncbi.nlm.nih.gov/pubmed/31450868
http://dx.doi.org/10.3390/ijms20174125
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author Xu, Wenjie
Fu, Wei
Zhu, Pengyu
Li, Zhihong
Wang, Chenguang
Wang, Chaonan
Zhang, Yongjiang
Zhu, Shuifang
author_facet Xu, Wenjie
Fu, Wei
Zhu, Pengyu
Li, Zhihong
Wang, Chenguang
Wang, Chaonan
Zhang, Yongjiang
Zhu, Shuifang
author_sort Xu, Wenjie
collection PubMed
description The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been widely applied in functional genomics research and plant breeding. In contrast to the off-target studies of mammalian cells, there is little evidence for the common occurrence of off-target sites in plants and a great need exists for accurate detection of editing sites. Here, we summarized the precision of CRISPR/Cas9-mediated mutations for 281 targets and found that there is a preference for single nucleotide deletions/insertions and longer deletions starting from 40 nt upstream or ending at 30 nt downstream of the cleavage site, which suggested the candidate sequences for editing sites detection by whole-genome sequencing (WGS). We analyzed the on-/off-target sites of 6 CRISPR/Cas9-mediated Arabidopsis plants by the optimized method. The results showed that the on-target editing frequency ranged from 38.1% to 100%, and one off target at a frequency of 9.8%–97.3% cannot be prevented by increasing the specificity or reducing the expression level of the Cas9 enzyme. These results indicated that designing guide RNA with high specificity may be the preferred factor to avoid the off-target events, and it is necessary to predict or detect off-target sites by WGS-based methods for preventing off targets caused by genome differences in different individuals.
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spelling pubmed-67471422019-09-27 Comprehensive Analysis of CRISPR/Cas9-Mediated Mutagenesis in Arabidopsis thaliana by Genome-Wide Sequencing Xu, Wenjie Fu, Wei Zhu, Pengyu Li, Zhihong Wang, Chenguang Wang, Chaonan Zhang, Yongjiang Zhu, Shuifang Int J Mol Sci Article The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been widely applied in functional genomics research and plant breeding. In contrast to the off-target studies of mammalian cells, there is little evidence for the common occurrence of off-target sites in plants and a great need exists for accurate detection of editing sites. Here, we summarized the precision of CRISPR/Cas9-mediated mutations for 281 targets and found that there is a preference for single nucleotide deletions/insertions and longer deletions starting from 40 nt upstream or ending at 30 nt downstream of the cleavage site, which suggested the candidate sequences for editing sites detection by whole-genome sequencing (WGS). We analyzed the on-/off-target sites of 6 CRISPR/Cas9-mediated Arabidopsis plants by the optimized method. The results showed that the on-target editing frequency ranged from 38.1% to 100%, and one off target at a frequency of 9.8%–97.3% cannot be prevented by increasing the specificity or reducing the expression level of the Cas9 enzyme. These results indicated that designing guide RNA with high specificity may be the preferred factor to avoid the off-target events, and it is necessary to predict or detect off-target sites by WGS-based methods for preventing off targets caused by genome differences in different individuals. MDPI 2019-08-23 /pmc/articles/PMC6747142/ /pubmed/31450868 http://dx.doi.org/10.3390/ijms20174125 Text en © 2019 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Xu, Wenjie
Fu, Wei
Zhu, Pengyu
Li, Zhihong
Wang, Chenguang
Wang, Chaonan
Zhang, Yongjiang
Zhu, Shuifang
Comprehensive Analysis of CRISPR/Cas9-Mediated Mutagenesis in Arabidopsis thaliana by Genome-Wide Sequencing
title Comprehensive Analysis of CRISPR/Cas9-Mediated Mutagenesis in Arabidopsis thaliana by Genome-Wide Sequencing
title_full Comprehensive Analysis of CRISPR/Cas9-Mediated Mutagenesis in Arabidopsis thaliana by Genome-Wide Sequencing
title_fullStr Comprehensive Analysis of CRISPR/Cas9-Mediated Mutagenesis in Arabidopsis thaliana by Genome-Wide Sequencing
title_full_unstemmed Comprehensive Analysis of CRISPR/Cas9-Mediated Mutagenesis in Arabidopsis thaliana by Genome-Wide Sequencing
title_short Comprehensive Analysis of CRISPR/Cas9-Mediated Mutagenesis in Arabidopsis thaliana by Genome-Wide Sequencing
title_sort comprehensive analysis of crispr/cas9-mediated mutagenesis in arabidopsis thaliana by genome-wide sequencing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747142/
https://www.ncbi.nlm.nih.gov/pubmed/31450868
http://dx.doi.org/10.3390/ijms20174125
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