A simple method for spray-on gene editing in planta

Abstract

Potential innovation in Plant research through the use of gene-edited and genetically modified plants iscurrently being hindered by inefficient and costly plant transformation. We show that naturally occurring carbon dots (quasi-spherical, <10nm nanoparticles) can act as a fast vehicle for carrying plasmids into mature plant cells, resulting in transient plant transformation in a number of important crop species with no negative impacts on photosynthesis or growth. We further show that GFP, Cas9, and gRNA introduced into wheat via foliar application (spraying on) of plasmid coated carbon dots are expressed and, in the case of Cas9, make genome edits in SPO11 genes. Therefore, we present a protocol for spray-on gene editing that is simple, inexpensive, fast, transforms in planta, and is applicable to multiple crop species. We believe this technique creates many opportunities for the future of plant transformation in research and shows great promise for plant protein production systems.Recent advances in plant biotech, particularly manipulation of photosynthesis, have shown the ability to obtain huge increases in plant efficiency and yield. For example, the RIPE project 1 obtained up to a 15% increase in biomass 2 and a ∼40% increase in productivity 3 by reducing photoprotection latency times and by avoiding photorespiration. These examples show the true power of GM -not only could these changes increase global food security (a growing issue with our population still increasing 4,5 , and climate change conferring multiple environmental stresses 6-8 ), but since these advances also increase the amount of carbon being fixed, it could have potential for also mitigating climate change 9,10 . This is important because, as noted, the effects of climate change exacerbate food insecurity further.Plant biotechnology can also enhance food security and biomass production by improving crop resistance to herbivores, pests, and environmental stresses. Additionally, GM techniques can enhance the nutritional value of the food produced, as seen with purple tomatoes 11,12 , enhancing the lipid content of oil crops to provide an alternative to dwindling fish oil stocks 13,14 and improving the quality of staple crops such as wheat 15,16 .However, the scope extends beyond edible compounds, as plant biotech is allowing the production of biofuels 17 , and has shown success producing pharmaceuticals 18 , including the efficient and speedy production of vaccines 19 . These advancements have been aided by new fields such as Synthetic biology, and gene editing tools becoming more versatile and useable.However, there is currently a significant bottleneck 20 limiting the potential application of these ideas and advances, and that is the cost, both in time and resources, of current plant transformation methods. All plant transformation must currently utilise either Agrobacterium tumefaciens 21 , biolistics 22 , or regeneration from PEG transformed protoplasts 23 , as a vehicle to introduce DNA, regardless of whether the changes are transient (n...

Differentiated impacts of human interventions on nature

“…For mutagens such as rDNA, the efficiency of delivery, that is, causing penetration of the target cell and relevant subcellular locations, is low, as is the frequency of its integration into chromosomes. Therefore, organisms to be transformed through the use of rDNA have to be concentrated and kept free of any contamination (Doyle et al, 2019). In many cases, from bacteria to plants, additional chemicals such as antibiotics are used to both concentrate and protect the rare recombinant cell.…”

“…Recognizing this, biotechnologists have been searching for tools that would be both unregulated and could be used outside of the laboratory (Doyle et al, 2019). In an example from the patent literature, the issue of scalability There is a need for introducing nucleic acids, such as DNA or RNA .…”

“…The ability to deliver the materials (DNA, RNA, proteins) needed to cause edits or silencing into cells has limited how quickly the technology can be applied. To transition from radiation and chemical mutagens to rDNA genetic engineering required finding ways to get the DNA into a cell, and where relevant the cell nucleus, intact and in a state that it could integrate or recombine with chromosomes (or replicate independently), and then regenerate an entire organism from a single transformed cell (Birch, 1997;Doyle et al, 2019).…”

Genetic Transformation in Peach (Prunus persica L.): Challenges and Ways Forward

“…In recent years, nanoparticles have been extensively utilized in many areas of research (reviewed by [ 90 ]). Successful nanoparticle-mediated introduction of DNA plasmids into plant cells at relatively high efficiencies has been demonstrated (e.g., [ 91 , 92 ]). The methodology is relatively simple and may offer certain advantages such as the absence of phytotoxicity and high target cell coverage.…”

Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing

“…In addition, nanoparticles-based RNP delivery has been shown to facilitate mammalian genome editing and can be potentially applied to plant, overcoming the transformation barrier from cell walls (Cunningham et al 2018). Most recently, various nanomaterials such as carbon dots (\ 10 nm) and carbon nanotubes have been demonstrated as a fast and simple method to deliver plasmids and possibly RNPs into various mature plant cells by spraying, leaf dipping, or leaf infiltration (Demirer et al 2019;Doyle et al 2019).…”