DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins

Abstract

The combined availability of whole genome sequences and genome editing tools is set to revolutionize the field of fruit biotechnology by enabling the introduction of targeted genetic changes with unprecedented control and accuracy, both to explore emergent phenotypes and to introduce new functionalities. Although plasmid-mediated delivery of genome editing components to plant cells is very efficient, it also presents some drawbacks, such as possible random integration of plasmid sequences in the host genome. Additionally, it may well be intercepted by current process-based GMO regulations, complicating the path to commercialization of improved varieties. Here, we explore direct delivery of purified CRISPR/Cas9 ribonucleoproteins (RNPs) to the protoplast of grape cultivar Chardonnay and apple cultivar such as Golden delicious fruit crop plants for efficient targeted mutagenesis. We targeted MLO-7, a susceptible gene in order to increase resistance to powdery mildew in grape cultivar and DIPM-1, DIPM-2, and DIPM-4 in the apple to increase resistance to fire blight disease. Furthermore, efficient protoplast transformation, the molar ratio of Cas9 and sgRNAs were optimized for each grape and apple cultivar. The targeted mutagenesis insertion and deletion rate was analyzed using targeted deep sequencing. Our results demonstrate that direct delivery of CRISPR/Cas9 RNPs to the protoplast system enables targeted gene editing and paves the way to the generation of DNA-free genome edited grapevine and apple plants.

Targeted genome editing inNicotiana tabacumusing inducible CRISPR/Cas9 system

“…In general, plant genome editing is conducted using the CRISPR/Cas9 system, in which the Cas9 gene is usually driven by a constitutive CaMV 35S promoter or plant native promoter [10,44,45,46]. However, constitutive expression of Cas9 is not necessary, given the fact that transient expression of Cas9 in plants is enough to induce the targeted mutagenesis [35,45,46].…”

“…In general, plant genome editing is conducted using the CRISPR/Cas9 system, in which the Cas9 gene is usually driven by a constitutive CaMV 35S promoter or plant native promoter [10,44,45,46]. However, constitutive expression of Cas9 is not necessary, given the fact that transient expression of Cas9 in plants is enough to induce the targeted mutagenesis [35,45,46]. Moreover, knockout of fundamentally significant genes (almost 10% of the Arabidopsis genome) would result in severe pleiotropic effects or lethality [47,48], and the lack of corresponding loss-of-function mutants makes it impossible to decipher the functions of these indispensable genes.…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…Using Cas9 RNA or protein presents the advantage of not having to work on optimizing expression by defining sequences codon-optimized for the host plant and selecting appropriate promoters. Preassembled complexes of purified Cas9 protein and guide RNA (RNPs) were transfected into protoplasts of Arabidopsis thaliana, Nicotiana attenuata, Petunia x hybrida, grapevine, apple, lettuce and rice [74,76,95] and bombarded into immature embryos of bread and durum wheat [73,96], and maize [75]. In all cases, RNPs showed good cleavage efficiency (4-46%), with (when tested) no detectable mosaicism, suggesting a very early action of the nuclease before the first cell division [74,76].…”

Crop genes modified using CRISPR/Cas system

“…Regenerated plants were not produced in the study. The presumed effect is increased resistance (Malnoy et al, 2016).…”