(174ae) Plant Genome Engineering with Nanotechnology for Agricultural Applications

Authors: 
Demirer, G. S., University of California
Zhang, H., University of California
Matos, J., University of California
Aditham, A., University of California
Staskawicz, B., University of California
Landry, M., Chan Zuckerberg Biohub
RESEARCH INTEREST
Food security is threatened by decreasing crop yields and increasing consumption in the light of climate change, population growth, and a shortage of arable land. 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 herbicides, insects, diseases, drought and heat. Despite the recent significant progress in genome editing, most plant species still remain difficult to genetically engineer. The two bottlenecks of generating engineered plants are (i) efficient biomolecule delivery into plant cells through the rigid cell wall and (ii) the regeneration of transformed tissues. The workhorse method of DNA delivery to plants, Agrobacterium, limits the range of transformable species and results in uncontrolled DNA integration, hence eliciting genetically modified organism (GMO) regulatory purview. In addition, it is currently not possible to edit plant germline cells, limiting our ability to engineer consumer-relevant crops. The development of a transformation tool that is (i) plant species-independent, (ii) non-pathogenic, (iii) non-integrating and/or DNA-free, and (iv) capable of transforming germline cells will greatly advance agricultural biotechnology. The unique and tunable physiochemical properties of nanomaterials can offer strategies for such desired plant genome engineering applications. Drawing on my interdisciplinary training, I aim to develop a multifaceted toolset using nanomaterials to provide food security for future generations.
Research Experience:
In my doctoral work, (i) I developed a nanomaterial-based gene delivery platform that efficiently delivers genes into agriculturally-relevant plants, without mechanical aid, in a non-toxic and non-integrating manner. I showed delivery of plasmid DNA to tobacco, arugula, cotton, and wheat leaves with engineered carbon nanotubes (CNTs), which resulted in strong transient expression of functional proteins. Importantly, I showed that exogenous DNA does not integrate into plant genome, relevant for avoiding GMO regulations. Next, (ii) I implemented this platform to deliver CRISPR plasmids targeting endogenous genes for knock-out. Through the transient expression of Cas9 and guide RNA in plant leaf cells, I obtained stable editing of target genes without transgene integration. My results have been patented by UC Berkeley and highlighted in C&EN, on NPR’s All Things Considered, and popular mechanics, among others.
In parallel, (iii) I developed a different nanoparticle surface chemistry for in planta delivery of small interfering RNA (siRNA) for DNA-free gene knock-down applications. The status quo for siRNA applications in plants involves biological delivery of a DNA coding for the siRNA. I demonstrated 95% gene silencing efficiency via direct delivery of siRNA with nanoparticles. I further verified that CNT-based delivery provides a significant delay in intracellular siRNA degradation, suggesting nanoparticles protect the cargo from nuclease degradation in addition to enabling cell wall transport. In a separate study, (iv) I systematically investigated the effect of nanomaterial parameters on plant cell uptake and gene silencing pathways. By leveraging the facile programmability of DNA nanostructures and origami, I elucidated the underlying principles of plant nanoparticle internalization and showed that plant endogenous gene silencing mechanisms can be altered by the nanostructure shape and the siRNA attachment locus. Finally, (v) I diversified my skillset with next-gen RNA sequencing to investigate the effect of nanomaterials on the plant transcriptome. Thus, armed with tools for efficient and rational biomolecule delivery into plants, I am excited to develop versatile strategies for the improvement of plant genome engineering.
Awards:
Faculty for the Future Fellowship, Schlumberger Foundation 2016-2020 ($50k/year, international)
MIT ChemE Rising Stars 2019
WCC Merck Research Award 2019
AIChE Women’s Initiative Committee Travel Award 2018
USDA National Institute of Food and Agriculture Award (Helped PI; Successful)
FFAR New Innovator Award (Helped PI; Successful)
USDA BBT Eager (Helped PI; Pending)
NSF EDGE CT (Helped PI; Pending)
AIChE 2018, Carbon Nanomaterials Graduate Student Award Session, 2nd place
AIChE 2017, Bionanotechnology Graduate Student Award Session, 3rd place
Selected Publications (4 of 12 total):
1. Demirer, G.S., Zhang, H., Matos, J. et al. High Aspect Ratio Nanomaterials Enable Delivery of Functional Genetic Material Without Transgenic DNA Integration in Mature Plants. Nature Nanotechnology (2019).
2. Demirer, G.S., Zhang, H., Goh, N. et al. Carbon Nanotube-Mediated DNA Delivery without Transgene Integration in Intact Plants. Nature Protocols (In press, 2019).
3. Demirer, G.S.*, Zhang, Hu.*, Zhang, Ho. et al. DNA Origami Nanostructure-Mediated Gene Silencing in Mature Plants. PNAS (2019).
4. Demirer, G.S., Zhang, H., Goh, N. at al. Carbon Nanocarriers Deliver siRNA to Intact Plant Cells for Efficient Gene Knockdown. ACS Nano (In review, 2019).
Future Research
The overarching goal of my lab will be to leverage chemical and biological engineering to create improved crops using new generation genome engineering tools. My lab will aim to address the critical limitations of: i) sub-optimal biomolecule delivery, (ii) inability to perform transgene or DNA-free editing, (iii) lack of tools for plant germline editing, and (iv) deficiency of plant protein interactions knowledge. Specifically, I will take three separate, initially independent, routes to address these challenges:
1) Developing transgene-free plant genome editing tools. Random integration of CRISPR DNA into plant genome causes off-target effects, endogenous gene disruption, and GMO-labeling. To avoid these complications and gain public acceptance of edited crops, researchers often go through the lengthy process of transgene segregation via breeding. However, for vegetatively propagated crops, transgene segregation in offspring is not possible. Thus, DNA-free genome editing approaches that do not cause integration will be transformative. One focus in my lab will be to develop new surface chemistries for the delivery of CRISPR ribonucleoproteins using nanoparticles that could be cleaved in planta via endogenous plant proteins. Completion of this goal will enable DNA-free genome editing for all plant species (including vegetatively propagated crops) without GMO regulation in many countries.
2) Discovering plant germline editing strategies. Another focus in my lab will be the application of gene editing technologies to plant species without available transformation methods, including wild relatives of major crops and orphan crops. Delivery of genome editing components to germline cells is a promising strategy to obtain gene-edited offspring in non-transformable species, which also has the advantage of avoiding arduous tissue regeneration. For this aim, my lab will first use the platform that I developed for CRISPR plasmid delivery. To increase the
germline editing efficiency, we will explore dual methods using geminivirus replication proteins and/or injection in tandem with nanoparticle carriers. We will also test different nanoparticle surface chemistries for optimal germline cell penetration. With the completion of aim 1), my lab will explore the delivery of ribonucleoproteins into germline cells of any desired plant species for stable and heritable editing without tissue regeneration.
3) Constructing plant genetic interaction maps. Plant polyploidy and genetic redundancy makes it difficult to find effective gene targets to produce desired traits. I will dedicate a subset of my lab’s efforts to understanding the extensive and complicated genetic networks controlling key agronomic traits and their interaction with environmental factors, initially focusing on stress resistance pathways, for which knowledge lags behind other traits. The main techniques for constructing plant genetic interaction maps in my lab will be stable knock-out of genes using CRISPR and transient knock-down of genes using RNAi. My lab’s elucidation of genetic interactions and agronomic traits will be aided by different “omics” approaches such as proteomics, metabolomics, and mass spectrometry-based screens, which will ultimately enable a comprehensive mapping of all pathways for a better handle on creating more robust crops.
In the long-term, I aim to incorporate findings from each of these tracks to address aforementioned challenges in plant genome engineering. The multidisciplinary nature of this proposed research combines chemical engineering, chemistry, plant biology, materials science, and computation, thus providing diverse training opportunities for doctoral and post-doctoral scholars.
TEACHING INTERESTS
My capability as a teacher arises from my undergraduate and graduate education, where I grew to appreciate the inherent multidisciplinary topics of Chemical Engineering coursework. As a UC Berkeley teaching assistant, I taught both core (Thermodynamics) and elective (Nanoscience and Engineering Biotechnology) classes, and also served as a team leader and teacher in the “Bay Area Scientists in Schools” program, where my team taught hands-on classes in public elementary schools about the environment. Additionally, I mentored four undergraduate research students and two high school students during my thesis studies; three of my mentees co-authored publications with me, and two of them went on to graduate school. I’ve learned an important lesson in my teaching and mentorship experiences: while it is rewarding to produce good science, my biggest impact will come from producing great scientists. Correspondingly, I maintain that teaching and mentorship – both in the classroom and in the laboratory – will be my most important contribution as a professor.
My initial experiences as a teacher at UC Berkeley have prominently contributed to my educational plan. I am enthusiastic to teach both core and elective courses, and interested in developing an interactive course to discuss the latest approaches in genome engineering and nanotechnology. Among the classes in my academic career, I have gained the most from the ones that encouraged critical thinking, interactive learning, and creative problem-solving, while drawing parallels to real-life scenarios. Therefore, I strive to implement such features in the courses I teach.