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Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane

Genome editing with sequence-specific nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is revolutionizing crop improvement. Developing efficient genome-editing protocols for highly polyploid crops, including sugarcane (x = 10–1...

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Autores principales: Eid, Ayman, Mohan, Chakravarthi, Sanchez, Sara, Wang, Duoduo, Altpeter, Fredy
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8525377/
https://www.ncbi.nlm.nih.gov/pubmed/34713257
http://dx.doi.org/10.3389/fgeed.2021.654996
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author Eid, Ayman
Mohan, Chakravarthi
Sanchez, Sara
Wang, Duoduo
Altpeter, Fredy
author_facet Eid, Ayman
Mohan, Chakravarthi
Sanchez, Sara
Wang, Duoduo
Altpeter, Fredy
author_sort Eid, Ayman
collection PubMed
description Genome editing with sequence-specific nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is revolutionizing crop improvement. Developing efficient genome-editing protocols for highly polyploid crops, including sugarcane (x = 10–13), remains challenging due to the high level of genetic redundancy in these plants. Here, we report the efficient multiallelic editing of magnesium chelatase subunit I (MgCh) in sugarcane. Magnesium chelatase is a key enzyme for chlorophyll biosynthesis. CRISPR/Cas9-mediated targeted co-mutagenesis of 49 copies/alleles of magnesium chelatase was confirmed via Sanger sequencing of cloned PCR amplicons. This resulted in severely reduced chlorophyll contents, which was scorable at the time of plant regeneration in the tissue culture. Heat treatment following the delivery of genome editing reagents elevated the editing frequency 2-fold and drastically promoted co-editing of multiple alleles, which proved necessary to create a phenotype that was visibly distinguishable from the wild type. Despite their yellow leaf color, the edited plants were established well in the soil and did not show noticeable growth retardation. This approach will facilitate the establishment of genome editing protocols for recalcitrant crops and support further optimization, including the evaluation of alternative RNA-guided nucleases to overcome the limitations of the protospacer adjacent motif (PAM) site or to develop novel delivery strategies for genome editing reagents.
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spelling pubmed-85253772021-10-27 Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane Eid, Ayman Mohan, Chakravarthi Sanchez, Sara Wang, Duoduo Altpeter, Fredy Front Genome Ed Genome Editing Genome editing with sequence-specific nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is revolutionizing crop improvement. Developing efficient genome-editing protocols for highly polyploid crops, including sugarcane (x = 10–13), remains challenging due to the high level of genetic redundancy in these plants. Here, we report the efficient multiallelic editing of magnesium chelatase subunit I (MgCh) in sugarcane. Magnesium chelatase is a key enzyme for chlorophyll biosynthesis. CRISPR/Cas9-mediated targeted co-mutagenesis of 49 copies/alleles of magnesium chelatase was confirmed via Sanger sequencing of cloned PCR amplicons. This resulted in severely reduced chlorophyll contents, which was scorable at the time of plant regeneration in the tissue culture. Heat treatment following the delivery of genome editing reagents elevated the editing frequency 2-fold and drastically promoted co-editing of multiple alleles, which proved necessary to create a phenotype that was visibly distinguishable from the wild type. Despite their yellow leaf color, the edited plants were established well in the soil and did not show noticeable growth retardation. This approach will facilitate the establishment of genome editing protocols for recalcitrant crops and support further optimization, including the evaluation of alternative RNA-guided nucleases to overcome the limitations of the protospacer adjacent motif (PAM) site or to develop novel delivery strategies for genome editing reagents. Frontiers Media S.A. 2021-04-29 /pmc/articles/PMC8525377/ /pubmed/34713257 http://dx.doi.org/10.3389/fgeed.2021.654996 Text en Copyright © 2021 Eid, Mohan, Sanchez, Wang and Altpeter. 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 Genome Editing
Eid, Ayman
Mohan, Chakravarthi
Sanchez, Sara
Wang, Duoduo
Altpeter, Fredy
Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane
title Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane
title_full Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane
title_fullStr Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane
title_full_unstemmed Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane
title_short Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane
title_sort multiallelic, targeted mutagenesis of magnesium chelatase with crispr/cas9 provides a rapidly scorable phenotype in highly polyploid sugarcane
topic Genome Editing
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8525377/
https://www.ncbi.nlm.nih.gov/pubmed/34713257
http://dx.doi.org/10.3389/fgeed.2021.654996
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