Genome Editing in Cowpea <i>Vigna unguiculata</i> Using CRISPR-Cas9
Cowpea (<i>Vigna unguiculata</i>) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cow...
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doaj-735b03db59284aec9f58314f2a80b75e2020-11-25T00:52:41ZengMDPI AGInternational Journal of Molecular Sciences1422-00672019-05-012010247110.3390/ijms20102471ijms20102471Genome Editing in Cowpea <i>Vigna unguiculata</i> Using CRISPR-Cas9Jie Ji0Chunyang Zhang1Zhongfeng Sun2Longlong Wang3Deqiang Duanmu4Qiuling Fan5State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, ChinaState Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, ChinaState Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, ChinaState Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, ChinaState Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, ChinaCollege of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, ChinaCowpea (<i>Vigna unguiculata</i>) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cowpea is an important legume crop, especially in many semi-arid countries. However, research in <i>Vigna unguiculata</i> is dramatically hampered by the lack of mutant resources and efficient tools for gene inactivation in vivo. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). We applied the CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the representative symbiotic nitrogen fixation (SNF) gene in <i>Vigna unguiculata</i>. Our customized guide RNAs (gRNAs) targeting symbiosis receptor-like kinase (SYMRK) achieved ~67% mutagenic efficiency in hairy-root-transformed plants, and nodule formation was completely blocked in the mutants with both alleles disrupted. Various types of mutations were observed near the PAM region of the respective gRNA. These results demonstrate the applicability of the CRISPR/Cas9 system in <i>Vigna unguiculata</i>, and therefore should significantly stimulate functional genomics analyses of many important agronomical traits in this unique crop legume.https://www.mdpi.com/1422-0067/20/10/2471genome editingCRISPR/Cas9cowpeasymbiosisnitrogen fixation |
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DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jie Ji Chunyang Zhang Zhongfeng Sun Longlong Wang Deqiang Duanmu Qiuling Fan |
spellingShingle |
Jie Ji Chunyang Zhang Zhongfeng Sun Longlong Wang Deqiang Duanmu Qiuling Fan Genome Editing in Cowpea <i>Vigna unguiculata</i> Using CRISPR-Cas9 International Journal of Molecular Sciences genome editing CRISPR/Cas9 cowpea symbiosis nitrogen fixation |
author_facet |
Jie Ji Chunyang Zhang Zhongfeng Sun Longlong Wang Deqiang Duanmu Qiuling Fan |
author_sort |
Jie Ji |
title |
Genome Editing in Cowpea <i>Vigna unguiculata</i> Using CRISPR-Cas9 |
title_short |
Genome Editing in Cowpea <i>Vigna unguiculata</i> Using CRISPR-Cas9 |
title_full |
Genome Editing in Cowpea <i>Vigna unguiculata</i> Using CRISPR-Cas9 |
title_fullStr |
Genome Editing in Cowpea <i>Vigna unguiculata</i> Using CRISPR-Cas9 |
title_full_unstemmed |
Genome Editing in Cowpea <i>Vigna unguiculata</i> Using CRISPR-Cas9 |
title_sort |
genome editing in cowpea <i>vigna unguiculata</i> using crispr-cas9 |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1422-0067 |
publishDate |
2019-05-01 |
description |
Cowpea (<i>Vigna unguiculata</i>) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cowpea is an important legume crop, especially in many semi-arid countries. However, research in <i>Vigna unguiculata</i> is dramatically hampered by the lack of mutant resources and efficient tools for gene inactivation in vivo. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). We applied the CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the representative symbiotic nitrogen fixation (SNF) gene in <i>Vigna unguiculata</i>. Our customized guide RNAs (gRNAs) targeting symbiosis receptor-like kinase (SYMRK) achieved ~67% mutagenic efficiency in hairy-root-transformed plants, and nodule formation was completely blocked in the mutants with both alleles disrupted. Various types of mutations were observed near the PAM region of the respective gRNA. These results demonstrate the applicability of the CRISPR/Cas9 system in <i>Vigna unguiculata</i>, and therefore should significantly stimulate functional genomics analyses of many important agronomical traits in this unique crop legume. |
topic |
genome editing CRISPR/Cas9 cowpea symbiosis nitrogen fixation |
url |
https://www.mdpi.com/1422-0067/20/10/2471 |
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