Agrobacterium- and a single Cas9-sgRNA transcript system-mediated high efficiency gene editing in perennial ryegrass

Abstract

Genome editing technologies provide a powerful tool for genetic improvement of perennial ryegrass, an important forage and turfgrass species worldwide. The sole publication for gene editing in perennial ryegrass used gene-gun for plant transformation and a dual promoter based CRISPR/Cas9 system for editing. However, their editing efficiency was low (5.9% or only one gene-edited plant produced). To test the suitability of the maize Ubiquitin 1 (<i>ZmUbi1</i>) promoter in gene editing of perennial ryegrass, we produced <i>ZmUbi1</i> promoter:<i>RUBY</i> transgenic plants. We observed that <i>ZmUbi1</i> promoter was active in callus tissue prior to shoot regeneration, suggesting that the promoter is suitable for Cas9 and sgRNA expression in perennial ryegrass for high-efficiency production of bi-allelic mutant plants. We then used the <i>ZmUbi1</i> promoter for controlling <i>Cas9</i> and sgRNA expression in perennial ryegrass. A ribozyme cleavage target site between the <i>Cas9</i> and sgRNA sequences allowed production of functional Cas9 mRNA and sgRNA after transcription. Using <i>Agrobacterium</i> for genetic transformation, we observed a 29% efficiency for editing the PHYTOENE DESATURASE gene in perennial ryegrass. DNA sequencing analyses revealed that most <i>pds</i> plants contained bi-allelic mutations. These results demonstrate that the expression of a single Cas9 and sgRNA transcript unit controlled by the <i>ZmUbi1</i> promoter provides a highly efficient system for production of bi-allelic mutants of perennial ryegrass and should also be applicable in other related grass species.