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Targeted mutagenesis in soybean using the CRISPR-Cas9 system

Genome editing is a valuable technique for gene function analysis and crop improvement. Over the past two years, the CRISPR-Cas9 system has emerged as a powerful tool for precisely targeted gene editing. In this study, we predicted 11 U6 genes in soybean (Glycine max L.). We then constructed two vec...

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Autores principales: Sun, Xianjun, Hu, Zheng, Chen, Rui, Jiang, Qiyang, Song, Guohua, Zhang, Hui, Xi, Yajun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4448504/
https://www.ncbi.nlm.nih.gov/pubmed/26022141
http://dx.doi.org/10.1038/srep10342
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author Sun, Xianjun
Hu, Zheng
Chen, Rui
Jiang, Qiyang
Song, Guohua
Zhang, Hui
Xi, Yajun
author_facet Sun, Xianjun
Hu, Zheng
Chen, Rui
Jiang, Qiyang
Song, Guohua
Zhang, Hui
Xi, Yajun
author_sort Sun, Xianjun
collection PubMed
description Genome editing is a valuable technique for gene function analysis and crop improvement. Over the past two years, the CRISPR-Cas9 system has emerged as a powerful tool for precisely targeted gene editing. In this study, we predicted 11 U6 genes in soybean (Glycine max L.). We then constructed two vectors (pCas9-GmU6-sgRNA and pCas9-AtU6-sgRNA) using the soybean U6-10 and Arabidopsis U6-26 promoters, respectively, to produce synthetic guide RNAs (sgRNAs) for targeted gene mutagenesis. Three genes, Glyma06g14180, Glyma08g02290 and Glyma12g37050, were selected as targets. Mutations of these three genes were detected in soybean protoplasts. The vectors were then transformed into soybean hairy roots by Agrobacterium rhizogenes infection, resulting in efficient target gene editing. Mutation efficiencies ranged from 3.2–9.7% using the pCas9-AtU6-sgRNA vector and 14.7–20.2% with the pCas9-GmU6-sgRNA vector. Biallelic mutations in Glyma06g14180 and Glyma08g02290 were detected in transgenic hairy roots. Off-target activities associated with Glyma06g14180 and Glyma12g37050 were also detected. Off-target activity would improve mutation efficiency for the construction of a saturated gene mutation library in soybean. Targeted mutagenesis using the CRISPR-Cas9 system should advance soybean functional genomic research, especially that of genes involved in the roots and nodules.
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spelling pubmed-44485042015-06-10 Targeted mutagenesis in soybean using the CRISPR-Cas9 system Sun, Xianjun Hu, Zheng Chen, Rui Jiang, Qiyang Song, Guohua Zhang, Hui Xi, Yajun Sci Rep Article Genome editing is a valuable technique for gene function analysis and crop improvement. Over the past two years, the CRISPR-Cas9 system has emerged as a powerful tool for precisely targeted gene editing. In this study, we predicted 11 U6 genes in soybean (Glycine max L.). We then constructed two vectors (pCas9-GmU6-sgRNA and pCas9-AtU6-sgRNA) using the soybean U6-10 and Arabidopsis U6-26 promoters, respectively, to produce synthetic guide RNAs (sgRNAs) for targeted gene mutagenesis. Three genes, Glyma06g14180, Glyma08g02290 and Glyma12g37050, were selected as targets. Mutations of these three genes were detected in soybean protoplasts. The vectors were then transformed into soybean hairy roots by Agrobacterium rhizogenes infection, resulting in efficient target gene editing. Mutation efficiencies ranged from 3.2–9.7% using the pCas9-AtU6-sgRNA vector and 14.7–20.2% with the pCas9-GmU6-sgRNA vector. Biallelic mutations in Glyma06g14180 and Glyma08g02290 were detected in transgenic hairy roots. Off-target activities associated with Glyma06g14180 and Glyma12g37050 were also detected. Off-target activity would improve mutation efficiency for the construction of a saturated gene mutation library in soybean. Targeted mutagenesis using the CRISPR-Cas9 system should advance soybean functional genomic research, especially that of genes involved in the roots and nodules. Nature Publishing Group 2015-05-29 /pmc/articles/PMC4448504/ /pubmed/26022141 http://dx.doi.org/10.1038/srep10342 Text en Copyright © 2015, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Sun, Xianjun
Hu, Zheng
Chen, Rui
Jiang, Qiyang
Song, Guohua
Zhang, Hui
Xi, Yajun
Targeted mutagenesis in soybean using the CRISPR-Cas9 system
title Targeted mutagenesis in soybean using the CRISPR-Cas9 system
title_full Targeted mutagenesis in soybean using the CRISPR-Cas9 system
title_fullStr Targeted mutagenesis in soybean using the CRISPR-Cas9 system
title_full_unstemmed Targeted mutagenesis in soybean using the CRISPR-Cas9 system
title_short Targeted mutagenesis in soybean using the CRISPR-Cas9 system
title_sort targeted mutagenesis in soybean using the crispr-cas9 system
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4448504/
https://www.ncbi.nlm.nih.gov/pubmed/26022141
http://dx.doi.org/10.1038/srep10342
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