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

Karolina Hus , Alexander Betekhtin , Artur Piński , Magdalena Rojek-Jelonek , Ewa Grzebelus , Candida Nibau , Mingjun Gao , Katja E. Jaeger , Glyn Jenkins , John H. Doonan , Robert Hasterok

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.
Author Karolina Hus - Uniwersytet Śląski
Karolina Hus,,
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, Alexander Betekhtin - Uniwersytet Śląski
Alexander Betekhtin,,
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, Artur Piński - Uniwersytet Śląski
Artur Piński,,
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, Magdalena Rojek-Jelonek - Uniwersytet Śląski
Magdalena Rojek-Jelonek,,
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, Ewa Grzebelus (FoBaH / Department of Plant Biology and Biotechnology)
Ewa Grzebelus,,
- Department of Plant Biology and Biotechnology
, Candida Nibau - Aberystwyth University
Candida Nibau,,
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, Mingjun Gao - University of Cambridge (CAM)
Mingjun Gao,,
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, Katja E. Jaeger - University of Cambridge (CAM), Leibniz Institute of Vegetable and Ornamental Crops
Katja E. Jaeger,,
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, Glyn Jenkins - Aberystwyth University
Glyn Jenkins,,
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, John H. Doonan - Aberystwyth University
John H. Doonan,,
-
et al.`
Journal seriesFrontiers in Plant Science, ISSN 1664-462X, (N/A 100 pkt)
Issue year2020
Vol11
Pages1-21
Publication size in sheets1
Article number614
Keywords in EnglishCRISPR/Cas9 system, targeted mutagenesis, Brachypodium distachyon, Brachypodium hybridum, Agrobacterium-mediated transformation, transient protoplast assay
ASJC Classification1110 Plant Science
DOIDOI:10.3389/fpls.2020.00614
URL https://www.frontiersin.org/articles/10.3389/fpls.2020.00614/pdf
Languageen angielski
LicenseJournal (articles only); author's original; Uznanie Autorstwa (CC-BY); after publication
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A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum of 22-05-2020
4,92 MB
Score (nominal)100
Score sourcejournalList
Publication indicators Scopus SNIP (Source Normalised Impact per Paper): 2018 = 1.328; WoS Impact Factor: 2018 = 4.106 (2) - 2018=4.855 (5)
Citation count*
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FinansowanieThis research was funded by the National Science Centre Poland (grant nos. DEC-2014/14/M/NZ2/00519 and DEC2018/31/N/NZ1/01418). JD, GJ, and CN were supported by BBSRCgrantBB/M009459/1.
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* presented citation count is obtained through Internet information analysis and it is close to the number calculated by the Publish or Perish system.
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