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

doi:10.3389/fpls.2020.00614

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

Front Plant Sci. 2020 May 20:11:614. doi: 10.3389/fpls.2020.00614. eCollection 2020.

Authors

Affiliations

¹ Plant Cytogenetics and Molecular Biology Group, Institute of Biology, Biotechnology and Environmental Protection, Faculty of Natural Sciences, University of Silesia in Katowice, Katowice, Poland.
² Department of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Cracow, Cracow, Poland.
³ National Plant Phenomics Centre, Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, United Kingdom.
⁴ Sainsbury Laboratory, University of Cambridge, Cambridge, United Kingdom.
⁵ Department for Plant Adaptation, Leibniz Institute of Vegetable and Ornamental Crops, Großbeeren, Germany.
⁶ Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, United Kingdom.

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.