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Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava

Cassava is the world’s most essential food root crop, generating calories to millions of Sub-Saharan African subsistence farmers. Cassava leaves and roots contain toxic quantities of the cyanogenic glycoside linamarin. Consumption of residual cyanogens results in cyanide poisoning due to conversion...

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Autores principales: Juma, Bicko Steve, Mukami, Asunta, Mweu, Cecilia, Ngugi, Mathew Piero, Mbinda, Wilton
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9644188/
https://www.ncbi.nlm.nih.gov/pubmed/36388608
http://dx.doi.org/10.3389/fpls.2022.1009860
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author Juma, Bicko Steve
Mukami, Asunta
Mweu, Cecilia
Ngugi, Mathew Piero
Mbinda, Wilton
author_facet Juma, Bicko Steve
Mukami, Asunta
Mweu, Cecilia
Ngugi, Mathew Piero
Mbinda, Wilton
author_sort Juma, Bicko Steve
collection PubMed
description Cassava is the world’s most essential food root crop, generating calories to millions of Sub-Saharan African subsistence farmers. Cassava leaves and roots contain toxic quantities of the cyanogenic glycoside linamarin. Consumption of residual cyanogens results in cyanide poisoning due to conversion of the cyanogens to cyanide in the body. There is a need for acyanogenic cassava cultivars in order for it to become a consistently safe and acceptable food, and commercial crop. In recent years, the CRISPR/Cas system, has proven to be the most effective and successful genome editing tool for gene function studies and crop improvement. In this study, we performed targeted mutagenesis of the MeCYP79D1 gene in exon 3, using CRISPR/Cas9, via Agrobacterium-mediated transformation. The vector design resulted in knockout in cotyledon-stage somatic embryos regenerated under hygromycin selection. Eight plants were recovered and genotyped. DNA sequencing analysis revealed that the tested putative transgenic plants carried mutations within the MeCYP79D1 locus, with deletions and substitutions being reported upstream and downstream of the PAM sequence, respectively. The levels of linamarin and evolved cyanide present in the leaves of mecyp79d1 lines were reduced up to seven-fold. Nevertheless, the cassava linamarin and cyanide were not completely eliminated by the MeCYP79D1 knockout. Our results indicate that CRISPR/Cas9-mediated mutagenesis is as an alternative approach for development of cassava plants with lowered cyanide content.
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spelling pubmed-96441882022-11-15 Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava Juma, Bicko Steve Mukami, Asunta Mweu, Cecilia Ngugi, Mathew Piero Mbinda, Wilton Front Plant Sci Plant Science Cassava is the world’s most essential food root crop, generating calories to millions of Sub-Saharan African subsistence farmers. Cassava leaves and roots contain toxic quantities of the cyanogenic glycoside linamarin. Consumption of residual cyanogens results in cyanide poisoning due to conversion of the cyanogens to cyanide in the body. There is a need for acyanogenic cassava cultivars in order for it to become a consistently safe and acceptable food, and commercial crop. In recent years, the CRISPR/Cas system, has proven to be the most effective and successful genome editing tool for gene function studies and crop improvement. In this study, we performed targeted mutagenesis of the MeCYP79D1 gene in exon 3, using CRISPR/Cas9, via Agrobacterium-mediated transformation. The vector design resulted in knockout in cotyledon-stage somatic embryos regenerated under hygromycin selection. Eight plants were recovered and genotyped. DNA sequencing analysis revealed that the tested putative transgenic plants carried mutations within the MeCYP79D1 locus, with deletions and substitutions being reported upstream and downstream of the PAM sequence, respectively. The levels of linamarin and evolved cyanide present in the leaves of mecyp79d1 lines were reduced up to seven-fold. Nevertheless, the cassava linamarin and cyanide were not completely eliminated by the MeCYP79D1 knockout. Our results indicate that CRISPR/Cas9-mediated mutagenesis is as an alternative approach for development of cassava plants with lowered cyanide content. Frontiers Media S.A. 2022-10-26 /pmc/articles/PMC9644188/ /pubmed/36388608 http://dx.doi.org/10.3389/fpls.2022.1009860 Text en Copyright © 2022 Juma, Mukami, Mweu, Ngugi and Mbinda https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Juma, Bicko Steve
Mukami, Asunta
Mweu, Cecilia
Ngugi, Mathew Piero
Mbinda, Wilton
Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava
title Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava
title_full Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava
title_fullStr Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava
title_full_unstemmed Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava
title_short Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava
title_sort targeted mutagenesis of the cyp79d1 gene via crispr/cas9-mediated genome editing results in lower levels of cyanide in cassava
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9644188/
https://www.ncbi.nlm.nih.gov/pubmed/36388608
http://dx.doi.org/10.3389/fpls.2022.1009860
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