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

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

doi:10.3389/fpls.2020.00614

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.

Highlights

Precise and efficient genome targeting technologies are invaluable for functional genomics
Single gRNA sequences were designed for fasciclin-like arabinogalactan protein (FLA) gene (BbsI gRNA) and CDKG1 gene (TaqI gRNA) and two gRNA sequences for phytoene desaturase (PDS) gene (XcmI gRNA, SalI gRNA), pectin methylesterase (PME) gene (BsrGI gRNA, AccI gRNA) and CDKG2 gene (BpmI gRNA and BsaWI gRNA) (Figure 4)
The transient protoplast assay was conducted on protoplasts of Bd21 leaves and, since the single nucleotide polymorphism (SNP) is in the “non-seed” gRNA region, the Cas9 endonuclease was able to introduce DSBs in the target gene

Summary

Introduction

Precise and efficient genome targeting technologies are invaluable for functional genomics. MGL + S50 + AS30 [500 mL]: 2.5 g tryptone + 1.25 g yeast extract + 2.6 g NaCl + 5 g mannitol + 1.16 g Lglutamine + 0.25 g KH2PO4 + 0.1 g MgSO4·7H2O + up to 500 mL H2O, adjust the pH with KOH to 7.2, add 5 g micro agar; autoclave, let the medium to cool down, add 1 μL 1 mg/mL biotin + 500 μL 50 mg/mL spectinomycin + 500 μL 30 mg/mL acetosyringone, pour ∼25 mL of medium into sterile Petri dishes, store at 4◦C

Results

Conclusion