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

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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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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.

Original languageEnglish
Article number614
Number of pages21
JournalFrontiers in Plant Science
Volume11
DOIs
Publication statusPublished - 20 May 2020

Keywords

  • Agrobacterium-mediated transformation
  • Brachypodium distachyon
  • Brachypodium hybridum
  • CRISPR/Cas9 system
  • targeted mutagenesis
  • transient protoplast assay

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