Nanotechnology to advance CRISPR-Cas genetic engineering of plants

Population growth has increased our food and energy consumption, and changes in our climate have decreased crop yields and productivity, thereby strongly threatening our future food and energy security. To mitigate these threats, genetic engineering of plants can be employed to create crops that have higher yields and nutritional value, and are resistant to biotic and abiotic stresses such as herbicides, diseases, drought, and heat. Engineered plants can also improve biosynthesis of valuable products, such as therapeutics and chemicals, and enable improved clean energy production from plant biomass. In the current era of CRISPR/Cas genome editing, the possibilities with plant genetic engineering are limitless. However, the two bottlenecks of generating engineered plants are (i) efficient biomolecule transport into plant cells through the rigid cell wall and (ii) the regeneration of transformed tissues to whole plants.

During my PhD in the Landry lab at UC Berkeley, I focused on addressing the first bottleneck of biomolecular cargo delivery to plants. Currently, we are mostly limited to biotic delivery methods that only work efficiently in select plant species, cause random transgene integration into the plant genome and face strict regulatory restrictions. Limited delivery methods, laborious plant regeneration process in tissue culture combined with the regulatory hurdles significantly slow down the reach of genetically engineered plants to the markets and make them very costly. An analysis in 2019 showed that the development of a genetically modified crop takes an average of 13 years of research and development at a cost of $136 million, demonstrating that there are significant research and regulatory hurdles to genetically engineering a sustainable future¹.

Motivated by this challenge, we addressed the plant biomolecule delivery problem by leveraging advantageous properties of nanoparticles, specifically single-walled carbon nanotubes (SWNTs). SWNTs are fully made of carbon and shaped into a long hallow cylinder. This nanocylinder of 1 nm in diameter and ~600 nm in length was optimally small enough to transport across the plant cell walls and at the same time had enough surface area to carry large biomolecules on it into the cells. Using SWNTs, we developed a tool to deliver plasmid DNA and small interfering RNA into the leaves of diverse plant species, and cargoes performed their biological functions with high efficiency^2–5. This platform also has the benefit of expressing or silencing plant genes without any transgene integration, which helps substantially with the regulation of these engineered plants.

After the publication of these results, our nanoparticle platform has attracted a lot of attention from plant researchers and industry. Therefore, we started to think in more detail about all the ways that nanotechnology can address critical challenges in plant genetic engineering. This led to an exciting collaborative project between our lab and the labs of Jenny Mortimer at LBL and Sue Rhee at Carnegie Science. In addition to our lab’s expertise in nanotechnology and cargo delivery, Mortimer and Rhee labs brought expertise in plant biology, genomics and genetic engineering. This collaboration has recently generated a Nature Nanotechnology perspective article, which is one of my papers that I am the proudest of⁶.

In this perspective article, we identified major barriers preventing CRISPR-mediated genetic engineering from reaching its full potential in plants, and we discussed the ways that nanoparticle technologies can lower or eliminate these barriers (Box 2 of the perspective paper). For instance, one crucial challenge is our inability to transform plant germline cells, and we hypothesized that the use of high tensile strength nanomaterials will be beneficial to transform pollen through large pollen surface apertures. We also suggested that the combined use of nanomaterials with other physical approaches such as microinjection for the transformation of flowers and shoot apical meristem is highly promising. Another technical limitation is the low efficiency of CRISPR homology directed repair (HDR) in plants. We proposed that the use of negatively charged nanoparticles to stabilize the Cas-guide RNA complex and carry a modified donor DNA interacting with Cas RNPs to shuttle the template to the nucleus could be a part of the solution. It could also help to perform time-staggered delivery of Cas protein, guide RNA, and donor DNA with nanoparticles for increasing HDR efficiencies in plants.

Furthermore, we highlighted some important outstanding questions pertaining to the nanomaterial-mediated CRISPR editing in plants (Box 3 of the perspective paper) to facilitate future discussion and creation of nanotechnologies for agriculture, bioproduct synthesis and bioenergy fields. One such outstanding question is “Would the regulation of gene edited plants using nanoparticles be different than traditionally edited plants?”. Another unknown is whether nanoparticles persist in downstream generations of edited plant offspring and what will be the environmental lifecycles and safety implications of nanomaterials on microbes and animals. We also need to start thinking about how to make these nanotechnologies widely available and routinely used in plant biotechnology applications.

I hope that our perspective article and this blog serve as an inspiration and guideline to the community to develop technologies for plant genetic engineering. There are various outstanding questions, which call for attention from a diverse set of researchers, industry, policy makers, stakeholders, and farmers for progress in nanomaterial-mediated plant genetic engineering. I believe, through multidisciplinary collaborations, we can address these questions, and we need to remember that the timely advancement of the application of CRISPR technologies in plant engineering is crucial for our ability to feed and sustain the growing human population under changing global climate.

References

1. Landry, M. P. & Mitter, N. How nanocarriers delivering cargos in plants can change the GMO landscape. Nat. Nanotechnol. 14, 512–514 (2019).
2. Demirer, G. S., Zhang, H., Goh, N. S., González-Grandío, E. & Landry, M. P. Carbon nanotube–mediated DNA delivery without transgene integration in intact plants. Nat. Protoc. 14, 2954–2971 (2019).
3. Demirer, G. S. et al. High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nat. Nanotechnol. 14, 456–464 (2019).
4. Demirer, G. S. et al. Carbon nanocarriers deliver siRNA to intact plant cells for efficient gene knockdown. Sci. Adv. 6, eaaz0495 (2020).
5. Demirer, G. S. & Landry, M. P. Efficient Transient Gene Knock-down in Tobacco Plants Using Carbon Nanocarriers. Bio-protocol 11, e3897–e3897 (2021).
6. Demirer, G. S. et al. Nanotechnology to advance CRISPR–Cas genetic engineering of plants. Nat. Nanotechnol. (2021) doi:10.1038/s41565-021-00854-y.