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 (ZmUbi1) promoter in gene editing of perennial ryegrass, we produced ZmUbi1 promoter:RUBY transgenic plants. We observed that ZmUbi1 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 ZmUbi1 promoter for controlling Cas9 and sgRNA expression in perennial ryegrass. A ribozyme cleavage target site between the Cas9 and sgRNA sequences allowed production of functional Cas9 mRNA and sgRNA after transcription. Using Agrobacterium for genetic transformation, we observed a 29% efficiency for editing the PHYTOENE DESATURASE gene in perennial ryegrass. DNA sequencing analyses revealed that most pds plants contained bi-allelic mutations. These results demonstrate that the expression of a single Cas9 and sgRNA transcript unit controlled by the ZmUbi1 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.

Strategies for delivery of CRISPR/Cas-mediated genome editing to obtain edited plants directly without transgene integration

“…For these applications, it may be beneficial to incorporate a component to discourage integration and/or to select against cells in which integration occurred. These are similar to the tactics for identifying non-transgenic plants described in the above section on full transgenic selection, such as barnase, slower growth of RUBY transgenic plants, and conditional toxicity of certain compounds when combined with a transgene; for example, the CodA transgene converts nontoxic 5-FOA into toxic 5-FU ( Koprek et al, 1999 ; Breyer et al, 2014 ; Oliveira et al, 2015 ; He et al, 2018 ; Aliaga-Franco et al, 2019 ; Bánfalvi et al, 2020 ; He and Zhao, 2020 ; He et al, 2021 ; Kumar et al, 2022 ). Such protocols offer a path to removing transgenic plants by selection against transgenes instead of laborious and costly screening by molecular methods.…”

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

“…If individual genes are edited too inefficiently, it makes later screening and identification doubly difficult and time-consuming, so it is also important not to express too many gRNAs when constructing editing vectors [ 137 ]. Although it is difficult to obtain higher-order mutants using a single editing vector, we can use crosses between stably inherited mutants of different genes to obtain multiple mutants [ 138 , 139 , 140 ]. In 2022, Mu et al created two different double mutants by targeting two genes with a single sgRNA and then obtained GmBIC quadruple mutants by hybridization.…”

Perspectives for reducing seed shattering in ryegrasses

“…On the other hand, genome editing could potentially also be used to validate candidate genes for seed shattering and to transfer reduced seed shattering into elite cultivars. However, there are only very few records successful genome editing in ryegrass so far (Grogg et al, 2019;Kumar et al, 2022;Zhang et al, 2020) and most other forage grass species lack adapted transformation and gene editing systems. In addition, the current regulation in Europe does not allow for using plants derived from genome editing in breeding programs.…”