Developing a new methodology for marker-free gene editing in grapevine

Dr. Laurent Deluc, Associate Professor, Department of Horticulture, Oregon State University

In March 2018, the U.S. Department of Agriculture (USDA) released a statement that they will not regulate plants modified through genome editing. Gene editing, the most popular being the CRISPR/Cas9 system, holds enormous promise for the development of accelerated breeding programs focused on the release of improved plant materials. A current significant focus is to identify grapevine material resistant or tolerant to extreme environmental conditions like drought, cold, and heat. Another research avenue is to generate new material resistant to major pests and diseases like Powdery and Downy Mildew and Botrytis. The wine grape industry may benefit from this federal decision to breed future elite cultivars more amenable to abiotic and biotic stresses. Unfortunately, the generation of transgenic plants is, in most cases, necessary to conduct the gene editing. This results in integrating bacterial-related sequences in the plant genome, making the engineered materials labeled Genetically Modified Organisms. Because of the GMO’s poor acceptance, there is a need to identify new methods to deliver gene-editing components to the plants without inserting “foreign DNA” in the targeted plant genome. The grapevine commodity will then fully benefit from the outstanding potential of gene-editing technology for genetic improvement.

Delivering a Ribonucleoprotein (RNP) like the protein Cas9 complexed to an RNA molecule to intact plant cells could be a very innovative solution for accepting the gene-editing technology for many crops. The RNP delivery has been tested for years in animal cells (Ramakrishna et al. 2014). In grapevine, initial studies to deliver the RNP are to date mostly limited to “naked” plant cells (protoplasts) (Malnoy et al. 2016). Unfortunately, grapevine plant regeneration from protoplasts has yet to be optimized. The physical delivery of the RNP to regenerable plant tissue (material from which you can regenerate a plant) will be significant to fully maximize gene editing applications for the grapevine. Though, the RNP complex will have to deal with a substantial barrier, the cell wall. The cell wall is an integral component of plant cells. It performs many essential functions, including, but not limited to, the shape and strength of cells, the protection against physical shocks, the control of cell expansion, the transport of substances between and across the cell, and a barrier between the interior cellular components and the external environment. A plant cell wall’s structure and composition are complex, tissue-specific, and vary over time, making a pretty complicated structure to be crossed. Nanoparticles were shown to deliver different molecules (DNA, RNA, and protein) in mammalian cells. In plant cells, their use in agriculture begins to gain more attention with relative success to deliver different molecules in normal plant cells containing cell walls.

The scientific community currently pays much attention to a particular class of nanoparticles, the Cell-wall Penetrating Peptide (CPP). Compared to other nanoparticles like gold or silicone particles, they tend to be much less cytotoxic to the plants. They can help deliver large cargo molecules like protein, DNA, and RNA (Numata et al. 2018). It consists of short peptide sequences (5 to 30 amino acid residues) that facilitate the cargo’s penetration through the cell membrane. Recent studies have demonstrated CPP’s efficacy to penetrate intact plant cells (Numata et al. 2018). The development of methodology for CPP-mediated delivery of a CRISPR/CAs9 system to a regenerable grapevine plant material will then be a game-changer because it will align with the pursuit of generating edited grapevine material that is GMO-free.

In our lab, we have developed two research projects to examine the efficacy of several CPPs to deliver the CRISPR/Cas9 complex. In our lab, we have generated transgenic lines that express the Green Fluorescent Protein (GFP) ectopically. As proof of concept, we tested the penetration efficiency of various CPPs to deliver a RNP to knock out the GFP gene expression. Preliminary results are promising, but they need reproducibility. We are currently designing experiments to improve the penetration efficiency rate of several tested CPPs.

Consideration and conclusions:
The discovery of CRISPR/Cas9 for more than ten years has dramatically opened new perspectives for advancing fundamental knowledge and genetic improvement in plant sciences and breeding. As a major crop in the U.S., Grapevine will benefit from its technology tool to develop innovative and accelerated breeding programs. Introducing new performance traits into existing cultivars is a major quest for the grapevine industry. The resistance to major fungal and bacterial pathogens is often cited as essential traits for which conventional breeding and traditional biotechnology (genetic transformation) may not be suitable. The development of a new methodology to create gene-edited material that is marker-free is an attractive alternative to time-consuming breeding programs. Likewise, the application of synthetic biology via precise gene editing is of particular value for crop production. One significant example for synthetic biology is the editing of DNA regions involved in inducing or repressing gene expression, the promoter region. Gene editing of promoter regions can generate plant materials for which the expression of multiple genes associated with a particular cellular process and/or a trait could be modulated at a given time. Chemically inducible systems are fundamental tools in modern agriculture with many potential uses to control plant growth. For better public acceptance, this will require the generation of scarless precise genome-edited material of the promoter regions. The public will likely accept an editing process that is transgene-free and does not affect plant proteins’ integrity.

This work is currently funded by the Oregon Wine Board, the Erath Family Foundation, and a forthcoming USDA-National Institute of Food and Agriculture award.

Literature Cited

Malnoy M, Viola R, Jung M-H, Koo O-J, Kim S, Kim J-S, Velasco R, Nagamangala Kanchiswamy C. 2016. DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins. Front Plant Sci 7:1904. DOI: 10.3389/fpls.2016.01904

Numata K, Horii Y, Oikawa K, Miyagi Y, Demura T, Ohtani M. 2018. Library screening of cell-penetrating peptide for BY-2 cells, leaves of Arabidopsis, tobacco, tomato, poplar, and rice callus. Scientific Reports 8:10966. DOI: 10.1038/s41598-018-29298-6

Ramakrishna S, Kwaku Dad A-B, Beloor J, Gopalappa R, Lee S-K, Kim H. 2014. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res 24:1020-1027. DOI: 10.1101/gr.171264.113

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