A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum

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A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum. / Hus, Karolina; Betekhtin, Alexander; Pinski, Artur; Rojek-Jelonek, Magdalena; Grzebelus, Ewa; Nibau, Candida; Gao, Mingjun; Jaeger, Katja E.; Jenkins, Glyn; Doonan, John H.; Hasterok, Robert.

In: Frontiers in Plant Science, Vol. 11, 614, 20.05.2020.

Research output: Contribution to journalArticle

Harvard

Hus, K, Betekhtin, A, Pinski, A, Rojek-Jelonek, M, Grzebelus, E, Nibau, C, Gao, M, Jaeger, KE, Jenkins, G, Doonan, JH & Hasterok, R 2020, 'A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum', Frontiers in Plant Science, vol. 11, 614. https://doi.org/10.3389/fpls.2020.00614

APA

Hus, K., Betekhtin, A., Pinski, A., Rojek-Jelonek, M., Grzebelus, E., Nibau, C., Gao, M., Jaeger, K. E., Jenkins, G., Doonan, J. H., & Hasterok, R. (2020). A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum. Frontiers in Plant Science, 11, [614]. https://doi.org/10.3389/fpls.2020.00614

Vancouver

Hus K, Betekhtin A, Pinski A, Rojek-Jelonek M, Grzebelus E, Nibau C et al. A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum. Frontiers in Plant Science. 2020 May 20;11. 614. https://doi.org/10.3389/fpls.2020.00614

Author

Hus, Karolina ; Betekhtin, Alexander ; Pinski, Artur ; Rojek-Jelonek, Magdalena ; Grzebelus, Ewa ; Nibau, Candida ; Gao, Mingjun ; Jaeger, Katja E. ; Jenkins, Glyn ; Doonan, John H. ; Hasterok, Robert. / A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum. In: Frontiers in Plant Science. 2020 ; Vol. 11.

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@article{445370cad5e242ce932a344817dbff84,
title = "A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum",
abstract = "The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.",
keywords = "Agrobacterium-mediated transformation, Brachypodium distachyon, Brachypodium hybridum, CRISPR/Cas9 system, targeted mutagenesis, transient protoplast assay",
author = "Karolina Hus and Alexander Betekhtin and Artur Pinski and Magdalena Rojek-Jelonek and Ewa Grzebelus and Candida Nibau and Mingjun Gao and Jaeger, {Katja E.} and Glyn Jenkins and Doonan, {John H.} and Robert Hasterok",
year = "2020",
month = may,
day = "20",
doi = "10.3389/fpls.2020.00614",
language = "English",
volume = "11",
journal = "Frontiers in Plant Science",
issn = "1664-462X",
publisher = "Frontiers Media",

}

RIS (suitable for import to EndNote) - Download

TY - JOUR

T1 - A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum

AU - Hus, Karolina

AU - Betekhtin, Alexander

AU - Pinski, Artur

AU - Rojek-Jelonek, Magdalena

AU - Grzebelus, Ewa

AU - Nibau, Candida

AU - Gao, Mingjun

AU - Jaeger, Katja E.

AU - Jenkins, Glyn

AU - Doonan, John H.

AU - Hasterok, Robert

PY - 2020/5/20

Y1 - 2020/5/20

N2 - The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.

AB - The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.

KW - Agrobacterium-mediated transformation

KW - Brachypodium distachyon

KW - Brachypodium hybridum

KW - CRISPR/Cas9 system

KW - targeted mutagenesis

KW - transient protoplast assay

UR - http://www.scopus.com/inward/record.url?scp=85085871596&partnerID=8YFLogxK

U2 - 10.3389/fpls.2020.00614

DO - 10.3389/fpls.2020.00614

M3 - Article

C2 - 32508865

AN - SCOPUS:85085871596

VL - 11

JO - Frontiers in Plant Science

JF - Frontiers in Plant Science

SN - 1664-462X

M1 - 614

ER -

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