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CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme

ABSTRACT. The CRISPR/Cas9 system is rapidly becoming the reagent of choice for targeted mutagenesis and gene editing in crop species. There are currently intense research efforts in the crop sciences to identify efficient CRISPR/Cas9 platforms to carry out targeted mutagenesis and gene editing proje...

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Autores principales: Michno, Jean-Michel, Wang, Xiaobo, Liu, Junqi, Curtin, Shaun J, Kono, Thomas JY, Stupar, Robert M
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033229/
https://www.ncbi.nlm.nih.gov/pubmed/26479970
http://dx.doi.org/10.1080/21645698.2015.1106063
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author Michno, Jean-Michel
Wang, Xiaobo
Liu, Junqi
Curtin, Shaun J
Kono, Thomas JY
Stupar, Robert M
author_facet Michno, Jean-Michel
Wang, Xiaobo
Liu, Junqi
Curtin, Shaun J
Kono, Thomas JY
Stupar, Robert M
author_sort Michno, Jean-Michel
collection PubMed
description ABSTRACT. The CRISPR/Cas9 system is rapidly becoming the reagent of choice for targeted mutagenesis and gene editing in crop species. There are currently intense research efforts in the crop sciences to identify efficient CRISPR/Cas9 platforms to carry out targeted mutagenesis and gene editing projects. These efforts typically result in the incremental tweaking of various platform components including the identification of crop-specific promoters and terminators for optimal expression of the Cas9 enzyme and identification of promoters for expression of the CRISPR guide RNA. In this report, we demonstrate the development of an online web tool for fast identification of CRISPR/Cas9 target loci within soybean gene models, and generic DNA sequences. The web-tool described in this work can quickly identify a high number of potential CRISPR/Cas9 target sites, including restriction enzyme sites that can facilitate the detection of new mutations. In conjunction with the web tool, a soybean codon-optimized CRISPR/Cas9 platform was designed to direct double-stranded breaks to the targeted loci in hairy root transformed cells. The modified Cas9 enzyme was shown to successfully mutate target genes in somatic cells of 2 legume species, soybean and Medicago truncatula. These new tools may help facilitate targeted mutagenesis in legume and other plant species.
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spelling pubmed-50332292016-10-19 CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme Michno, Jean-Michel Wang, Xiaobo Liu, Junqi Curtin, Shaun J Kono, Thomas JY Stupar, Robert M GM Crops Food Research Papers ABSTRACT. The CRISPR/Cas9 system is rapidly becoming the reagent of choice for targeted mutagenesis and gene editing in crop species. There are currently intense research efforts in the crop sciences to identify efficient CRISPR/Cas9 platforms to carry out targeted mutagenesis and gene editing projects. These efforts typically result in the incremental tweaking of various platform components including the identification of crop-specific promoters and terminators for optimal expression of the Cas9 enzyme and identification of promoters for expression of the CRISPR guide RNA. In this report, we demonstrate the development of an online web tool for fast identification of CRISPR/Cas9 target loci within soybean gene models, and generic DNA sequences. The web-tool described in this work can quickly identify a high number of potential CRISPR/Cas9 target sites, including restriction enzyme sites that can facilitate the detection of new mutations. In conjunction with the web tool, a soybean codon-optimized CRISPR/Cas9 platform was designed to direct double-stranded breaks to the targeted loci in hairy root transformed cells. The modified Cas9 enzyme was shown to successfully mutate target genes in somatic cells of 2 legume species, soybean and Medicago truncatula. These new tools may help facilitate targeted mutagenesis in legume and other plant species. Taylor & Francis 2015-10-19 /pmc/articles/PMC5033229/ /pubmed/26479970 http://dx.doi.org/10.1080/21645698.2015.1106063 Text en © 2015 The Author(s). Published with license by Taylor & Francis Group, LLC http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License http://creativecommons.org/licenses/by-nc/3.0/, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
spellingShingle Research Papers
Michno, Jean-Michel
Wang, Xiaobo
Liu, Junqi
Curtin, Shaun J
Kono, Thomas JY
Stupar, Robert M
CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme
title CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme
title_full CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme
title_fullStr CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme
title_full_unstemmed CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme
title_short CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme
title_sort crispr/cas mutagenesis of soybean and medicago truncatula using a new web-tool and a modified cas9 enzyme
topic Research Papers
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033229/
https://www.ncbi.nlm.nih.gov/pubmed/26479970
http://dx.doi.org/10.1080/21645698.2015.1106063
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