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Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba
Although the CRISPR/Cas9 system has been successfully used for crop breeding, its application remains limited in forest trees. Here, we describe an efficient gene editing strategy for hybrid poplar, (Populus tremula × alba INRA clone 717-1B4) based on the Golden Gate MoClo cloning. To test the syste...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8597961/ https://www.ncbi.nlm.nih.gov/pubmed/33960379 http://dx.doi.org/10.1093/treephys/tpab066 |
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author | Triozzi, Paolo M Schmidt, Henry W Dervinis, Christopher Kirst, Matias Conde, Daniel |
author_facet | Triozzi, Paolo M Schmidt, Henry W Dervinis, Christopher Kirst, Matias Conde, Daniel |
author_sort | Triozzi, Paolo M |
collection | PubMed |
description | Although the CRISPR/Cas9 system has been successfully used for crop breeding, its application remains limited in forest trees. Here, we describe an efficient gene editing strategy for hybrid poplar, (Populus tremula × alba INRA clone 717-1B4) based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two single guide RNAs (sgRNAs) were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate the SHORT ROOT (SHR) gene. Moreover, we also tested its efficiency for introducing mutations in two genes simultaneously by expressing one sgRNA targeting a single site of the YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target the LBD12 and LBD4 genes simultaneously, using an independent construct. We generated hairy roots by Agrobacterium rhizogenes-mediated leaf transformation. Sequencing results confirmed the CRISPR/Cas9-mediated mutation in the targeted sites of PtaSHR. Biallelic and homozygous knockout mutations were detected. A deletion spanning both target sites and small insertions/deletions were the most common mutations. Out of the 22 SHR alleles sequenced, 21 were mutated. The phenotype’s characterization showed that transgenic roots with biallelic mutations for the SHR gene lacked a defined endodermal single cell layer, suggesting a conserved gene function similar to its homolog in Arabidopsis Arabidopsis thaliana (L.) Heynh. Sequencing results also revealed the high efficiency of the system for generating double mutants. Biallelic mutations for both genes in the yuc4/plt1 and lbd12/lbd4 roots were detected in three (yuc4/plt1) and two (lbd12/lbd4) out of four transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site was the most common mutations. This CRISPR/Cas9 strategy arises as a rapid, simple and standardized gene-editing tool to evaluate the gene role in essential developmental programs such as radial cell differentiation of poplar roots. |
format | Online Article Text |
id | pubmed-8597961 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-85979612021-11-18 Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba Triozzi, Paolo M Schmidt, Henry W Dervinis, Christopher Kirst, Matias Conde, Daniel Tree Physiol Methods Paper Although the CRISPR/Cas9 system has been successfully used for crop breeding, its application remains limited in forest trees. Here, we describe an efficient gene editing strategy for hybrid poplar, (Populus tremula × alba INRA clone 717-1B4) based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two single guide RNAs (sgRNAs) were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate the SHORT ROOT (SHR) gene. Moreover, we also tested its efficiency for introducing mutations in two genes simultaneously by expressing one sgRNA targeting a single site of the YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target the LBD12 and LBD4 genes simultaneously, using an independent construct. We generated hairy roots by Agrobacterium rhizogenes-mediated leaf transformation. Sequencing results confirmed the CRISPR/Cas9-mediated mutation in the targeted sites of PtaSHR. Biallelic and homozygous knockout mutations were detected. A deletion spanning both target sites and small insertions/deletions were the most common mutations. Out of the 22 SHR alleles sequenced, 21 were mutated. The phenotype’s characterization showed that transgenic roots with biallelic mutations for the SHR gene lacked a defined endodermal single cell layer, suggesting a conserved gene function similar to its homolog in Arabidopsis Arabidopsis thaliana (L.) Heynh. Sequencing results also revealed the high efficiency of the system for generating double mutants. Biallelic mutations for both genes in the yuc4/plt1 and lbd12/lbd4 roots were detected in three (yuc4/plt1) and two (lbd12/lbd4) out of four transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site was the most common mutations. This CRISPR/Cas9 strategy arises as a rapid, simple and standardized gene-editing tool to evaluate the gene role in essential developmental programs such as radial cell differentiation of poplar roots. Oxford University Press 2021-05-07 /pmc/articles/PMC8597961/ /pubmed/33960379 http://dx.doi.org/10.1093/treephys/tpab066 Text en © The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Methods Paper Triozzi, Paolo M Schmidt, Henry W Dervinis, Christopher Kirst, Matias Conde, Daniel Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba |
title | Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba |
title_full | Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba |
title_fullStr | Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba |
title_full_unstemmed | Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba |
title_short | Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba |
title_sort | simple, efficient and open-source crispr/cas9 strategy for multi-site genome editing in populus tremula × alba |
topic | Methods Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8597961/ https://www.ncbi.nlm.nih.gov/pubmed/33960379 http://dx.doi.org/10.1093/treephys/tpab066 |
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