Progress 04/01/19 to 03/31/22
Outputs
Target Audience:The target audience of our project is the plant science and biotechnology community, as we are developing tools for the delivery of nucleic acids to plants. Specifically, we are developping tools for the delivery of DNA, RNA, and proteins to plants in a plant species independent manner, and to enable genome editing in agriculturally relevant crops. Changes/Problems:Our project goals initially included the delivery of DNA vectors coding for CRISPR plasmids with PEI-SWNT, however, our recent publications found that this system was toxic to plant cells at concentrations needed for efficacious genome editing (Grandio et al. Journal of Nanobiotechnology 2021) and were unable to load and deliver large plasmids needed for genome editing applications (Zahir et al. ACS applied nanomaterials 2022). Therefore, we shifted to the delivery of mRNA and sgRNA cargoes with nanotubes, which was the focus of the research described in this terminal report. What opportunities for training and professional development has the project provided?This project has supported a graduate student and postdoc (both on fellowship for stipend support) in learning to develop experimental protocols, learning to perform data anlysis, and disseminating data in the form of conference presentations, and journal articles. Students and postdocs have cross-trained in nanoscience and plant biology, providing an interdisciplinary setting for career development. More broadly,I have mentored numerous trainees in my lab: 12 postdocs, 19 graduate students, and over 30 undergraduate researchers. As part of my mentoring practices, I provide professional development assessments for all of my mentees through individual development plans (IDPs), effective communication workplans, and career development worksheets, tools which I disseminate on my lab website. How have the results been disseminated to communities of interest?We have published over 1 dozen peer reviewed publications and given 98 talks on the contents of our research, which we are unable to list due to space limitations in the reporting template. What do you plan to do during the next reporting period to accomplish the goals?In summary, we have demonstrated that SWNTs can be successfully and efficiently functionalized with sgRNA or crRNA/tracrRNA while retaining integrity and Cas9in vitroactivity. We also demonstrated that gRNA can be desorbed from SWNTs in Plant cell lysate and thatin vivogene editing efficiency can be successfully accessed via amplicon sequencing. We are looking forward to deploying this technology forin vivogene editing in different plant tissues and species and under different conditions. Gene editing CRISPR-Cas technologies have revolutionized plant genetic engineering by allowing precise gene editing in many plant species. However, there are important limitations and bottlenecks that yet need to be addressed in order for this technology to have a broad sustainable impact in agriculture. Current plant transformation methods require delivery of gene editing reagents via Agrobacterium-mediated or Biolistic methods, which leads to random genome integration of Cas9 and gRNA gene cassettes. Transfected or bombarded plant tissues need then laborious tissue culture and antibiotic/herbicide selection to regenerate gene-edited plants that yet need to crossed and have progenies subsequently screened so that the transgene cassette can be segregated out and eliminated from the genome. Our work establishes the foundation for the delivery of Cas9 gene-editing reagents with nanoparticles to address these limitations by providing an alternative delivery avenue that is species-independent for transgene-free gene editing. These will be the areas of focus for our future work beyond the timescale of our grant. Our Ongoing efforts beyond this project include: (1) Testing different conditions for the preparation of SWNT-RNA suspensions in order to identify parameters leading to optimal loading capacity and integrity of gRNA (sgRNA, crRNA and tracrRNA); (2) Employing the use of Bioanalyzer to access gRNA integrity, quality and concentration of adsorbed vs non-adsorbed gRNAs onto SWNTs; (3) Characterizing of SWNT-mediated protection of loaded gRNAs against RNAse degradationin vitroandin vivo; and (4) Delivering SWNT-sgRNA (and SWNT-crRNA/tracrRNA) to Cas9-expressing plant materials - isolated protoplasts and intact plant tissues - and assessment of gene editing efficiency via amplicon sequencing. We have successfully established a pipeline for amplicon sequencing that will allow us to investigate gene editing efficiency in a large scale and to test different delivery methods, different concentrations of SWNT suspensions, targeting different plant tissues at different developmental stages, as well as in different species. We are currently testing delivery andin vivogene editing inN. Benthamianaroots, leaves, seedlings, and adult plants, Tomato plants, Maize plantlets, and Barley and Sorghum immature embryos and calli. Furthermore, ourin vivowork and gene editing assessments will also allow us to investigate the effects of plant growth conditions such as temperature and nutritional status on gene editing efficiency. One area we are also currently investigating is the impact of modulating cell wall composition and elasticity with drugs and buffers in a transient manner with the goal of increasing cargo delivery mediated by SWNTs but also additional nanoparticle carriers.?
Impacts
What was accomplished under these goals?
In our award period, we determined that the originally-planned materials, PEI-SWNT, were ineffective for genome editing applications. Instead, in this work, we tested several varieties of RNA used for gene editing with Cas9: single-guide RNA (sgRNA), CRISPR RNA (crRNA), and trans-activating CRISPR RNA (tracrRNA). crRNA and tracrRNA are the dual RNA components discovered in the wildtype Cas9 system, wherein crRNA (36nt) contains the target information and tracrRNA (67nt) contains the structural motif necessary for binding crRNA to Cas9. sgRNA (100 nt) is a synthetic chimera of the crRNA and tracrRNA and contains both the target sequence and the structural motif required to bind Cas9. RNA is purchased commercially (from IDT) as a synthetic oligonucleotide with chemical modifications to protect against environmental exoribonucleases, which greatly simplifies handling and storage. RNA-SWNTs are prepared as described previously for siRNA SWNT suspensions (Demirer, G.S. et al,Bio Protocols, 2021) ; we note that all three RNA types surveyed in this work were successful at suspending SWNT with identical preparation protocols. 50-500 ug of RNA are combined with raw SWNTs in 100 mM NaCl at a 2:1 mass ratio of RNA:SWNT and probe-tip sonicated for 30 minutes. The raw suspension is then centrifuged at high speed (15000-20000 rcf) for 60 minutes to pellet unsuspended SWNT bundles as well as amorphous carbon and residual catalyst leftover from SWNT synthesis. Approximately 40-60% of the total RNA is adsorbed onto individual SWNTs, and the remaining RNA free in solution is removed via 7-10 spin filtration steps through a 100K MWCO cellulosic membrane that allows free RNA to pass through with minimal loss of suspended SWNTs. A successful SWNT suspension is initially verified by eye, as colloidal SWNTs should appear as a homogenous gray/black liquid (Fig 1B). A stable RNA-SWNT suspension is further confirmed by near-infrared (nIR) fluorescence spectrometry, wherein strong nIR emission indicates the presence of individually suspended SWNTs (as bundled SWNT aggregates are highly quenched). Furthermore, we note the stability of RNA-SWNTs over time, as indicated by no loss in nIR fluorescence emission over the course of two months when stored at 4oC. sgRNA, crRNA, and tracrRNA suspend SWNTs to a high concentration (50-100 mg/L SWNT in 1 mL) and with reproducible yield similar to DNA-SWNTs; an important finding, as the cost of synthetic RNA oligonucleotides at the scale required for SWNT conjugation greatly exceeds the cost of DNA oligonucleotides of similar length. We hypothesize that either unfiltered RNA-SWNT (free RNA + adsorbed RNA) or filtered RNA-SWNT (adsorbed RNA, only) could function as an effective conjugate for delivery applications, so samples were collected before and after filtration for use in downstream assays. As the types of guide RNA used in this work (36-100nts) are longer than siRNAs used previously (20-25nts), and also require folding into a secondary structure to bind Cas9, we first sought to measure if the guide RNA structural integrity is maintained after ultrasonication with SWNTs. In order to assay the functionality of sgRNA, crRNA, and tracrRNA suspended onto SWNTs, we performed Cas9in vitroassays. In this assay SpCas9 protein is mixed with SWNT-RNA suspensions (both unfiltered and filtered), Cas9 Buffer (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9), PCR template DNA containing the CRISPR guide RNA target sequence, and incubated at 37oC for 8 hours. Cas9 mixed with free sgRNA or crRNA/tracrRNA were used as positive controls and Cas9 alone (no sgRNA, crRNA and tracrRNA) were used as negative controls. Functional RNPs (Cas9 complexed with guide RNA) are able to cleave the template DNA (757bp) and when run on agarose gel two fragments can be observed (440 and 313bp), while uncleaved DNA template indicates an absence of RNPin vitroactivity. When incubated with Cas9 Buffer, unfiltered SWNT-RNA suspensions led to template DNA cleavage while filtered suspensions did not suggesting that free (unbound) sgRNA or crRNA/tracrRNA in the unfiltered SWNT suspensions serve as the source for assembly of functional RNPs and cleavage of the template DNA. Importantly, the Cas9in vitroactivity confirms that sgRNA, crRNA and tracrRNA remain intact and functional after our suspension protocols, and confirm the ability of SWNT ability to deliver sgRNAs in a plant-relevant condition. Next, we investigated Cas9in vitroactivity for SWNT suspensions in the presence of Plant Cell Lysate. Upon introduction to a plant cell, RNA must desorb from the SWNT surface in order to bind Cas9 and trigger targeted cleavage in the host genome. We hypothesize that proteins and other biomolecules present in a complex biofluid could promote guide RNA (sgRNA, crRNA/tracrRNA) desorption and lead to assembly of functional RNPs that would then successfully cleave template DNA. Figure 3B shows that unfiltered SWNT suspensions led to template DNA cleavage when incubated with Cas9 in Cas9 Buffer or Plant Cell Lysate. As for filtered SWNT suspensions, while incubation in Cas9 Buffer did not lead to template DNA cleavage, incubation in Plant cell lysate led to template DNA cleavage indicating that sgRNA and crRNA/tracrRNA can be desorbed by cellular contents and lead to assembly of functional RNPs. To further investigate the desorption of gRNA through the formation of a biomolecular corona, wherein adsorbed RNA undergoes competitive binding with other molecules that have some affinity to the SWNT surface, we utilized a fluorescence-based assay previously developed in our lab (Pinals, R.L., Yang, D., et al.Ang. Chem., 2020) to measure the extent of RNA desorption when incubated in plant cell lysate containing a complex mixture of proteins and other biomolecules. In this assay, fluorescently-labeled ATTO550-tracrRNA was assembled with SWNTs with the resulting suspension showing no ATTO550 emission signal due to strong quenching caused by proximity to the nanotube surface. However, upon incubation in plant cell lysate fromNicotiana benthamianaprotoplasts, ATTO550 signal is recovered due to RNA desorption from SWNTs promoted by competitive protein adsorption. Compared to a control protein fibrinogen, which is known to promote desorption of nucleic acids from SWNTs (Pinals, R.L., Yang, D., et al.Ang. Chem., 2020), ATTO550-tracrRNA-SWNTs show a similar dose-dependent response in plant cell lysate, suggesting that complex biofluids such as those found inside plant cells would promote a significant proportion of RNA to become free for binding to Cas9. Furthermore, we found that the nIR fluorescence emission spectra of sgRNA-SWNTs exhibit a strong turn-on response in the presence of purified Cas9 protein. Due to intrinsic quenching from surface interactions between SWNTs and aromatic nucleic acid groups, nIR fluorescence turn-on of DNA/RNA-SWNTs in response to an analyte is a phenomena that is often suggestive of partial desorption of oligonucleotides adsorbed to the nanotube surface. Thus, the strong turn-on response of sgRNA-SWNTs in response to Cas9 suggests that Cas9 itself, in addition to endogenous proteins in the plant cell, could promote desorption of RNA from SWNTs.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Zhang, H.* Goh, N.S.*, Wang, J., Demirer, G.S., Butrus, S., Park, S-J, Landry, M.P.! Nanoparticle Cellular Internalization is Not Required for RNA Delivery to Mature Plant Leaves. Nature Nanotechnology (2022)
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2022
Citation:
Wang, J.W., Goh, N.S., Lien, E.S., Gonzales-Grandio, E., Landry, M.P.! Quantification of cell penetrating peptide mediated delivery of proteins in plant leaves. bioRxiv (2021)
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Zahir, A., Serag, M.F., Demirer, G.D., Torre, B., di Fabrizio, E., Landry, M.P., Habuchi, S.!, Mahfouz, M.! DNA-Carbon Nanotube Binding Mode Determines the Efficiency of Carbon Nanotube-Mediated DNA Delivery to Intact Plants. ACS Applied Nano Materials (2022)
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Ouassil, N., Pinals, R.L., ODonnell, J.T., Wang, J., Landry, M.P.! Supervised Learning Model Predicts Protein Adsorption to Nanotubes. Science Advances (2022)
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Jackson, C.T., Wang, J.W., Gonzalez-Grandio, E., Goh, N.S., Mun, J., Krishnan, S., Landry, M.P.! Polymer-Conjugated Carbon Nanotubes for Biomolecule Loading. ACS Nano (2021)
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Progress 04/01/21 to 03/31/22
Outputs
Target Audience:The target audience are researchers in plant biology and biotechnology, and synthetic biology. Our work targets a biotechnology, agriculture, and plant biology research community. We have published several papers in the reporting period (including protocols that can be followed by our colleagues), presented at conferences, and participated in panels and workshops. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The Landry Lab focuses on maintaining an open stream of communication between people and between disciplines to optimize scientific productivity. The Landry lab's first and to-date only PhD graduates are both principal investigators: Abraham Beyene leads the Beyene Lab at HHMI's Janelia Research Campus, and Gozde Demirer will start the Demirer Lab in the Caltech department of Chemical Engineering in 2022 - both of these students supported by NIFA. I work closely with each undergraduate or graduate student to identify a research area and project for the student to undertake in the Landry Lab, and work with students to ensure their thesis research plan combines an appropriately lengthy research goal (4-5 year project) while detailing key goals in a 6-month increment that may form stand-alone publications. I also enable students to work on a secondary collaborative project that ties in with an existing student or postdoc project in the group, to increase their peer-to-peer mentorship skills and instruct students on how to perform interdisciplinary science. Collaborations with other groups are highly encouraged as an opportunity to gain scientific breadth, and refine interpersonal and professional networking skills. In addition to publications, in 4-5th year of their doctoral work, my students are expected to work closely with me to write grants, and communicate with collaborators in the composition of those grants. My students are also expected to occasionally serve as graduate student instructors (TAs) to tune their teaching skills. My students also present their work at conferences tied to their research topics at least annually, and also give research presentation updates within the group and within the department multiple times per year. The Landry lab also maintains a list of fellowship opportunities for domestic and international students alike (http://landrylab.com/fellowships/), and an active resource for mental health (http://landrylab.com/resources-for-mental-health/). Additionally, because professional development is a key aspect of undergraduate, graduate, and postdoctoral education, I perform annual individual development plan (IDP) assessments, annual diversity, equity and inclusion workshops, annual mentor-mentee relationship assessments, and also standardized performance assessments and reporting templates for undergraduate researchers (http://landrylab.com/professional-development/). How have the results been disseminated to communities of interest?The Landry Lab has published a series of peer reviewed publications in 2020-2021 to disseminate our results, as follows: Wang, J.W., Cunningham, F.J., Goh, N., Boozarpour, N.N., Pham, M., Landry, M.P.‡ Nanoparticles for protein delivery in planta. Current Opinion in Plant Biology (2021). 60(102052). Wang, J., Cunningham, F. J., Goh, N., Boozarpour, N.N., Pham, M., Landry, M.P.‡ A nanoparticle approach for protein delivery in planta. Current Opinion in Plant Biology (2021) Demirer, G.S.‡, Silva, T.N., Jackson, C.T., Thomas, J.B., Ehrhardt, D., Rhee, S.Y.‡, Mortimer, J.C.‡, Landry, M.P.‡ Nanotechnology to advance CRISPR/Cas genetic engineering of plants. Nature Nanotechnology (2021). Demirer, G.S.‡, Landry, M.P.‡ Efficient Transient Gene Knock-down in Tobacco Plants Using Carbon Nanocarriers. Bio-protocol (2021). 11(1) Hofmann, T., Lowry‡, G.V.‡, Ghoshal, S., Tufenkji, N., Brambilla, D., Dutcher, J.R., Gilbertson, L.M., Giraldo, J. P., Kinsella, J. M., Landry, M.P., Lovell, W., Naccache, R., Paret, M., Pedersen, J. A., Unrine, J., M., White, J.C., Wilkinson, K.J. Technology readiness and overcoming barriers to sustainably implement nanotechnology-enabled plant agriculture. Nature Food (2020). DOI: 10.1038/s43016-020-0110-1 Zhang, H., Zhang, H., Demirer, G.S., Gonzales-Grandio, E., Fan, C., Landry, M.P.‡ Engineering DNA nanostructures for siRNA delivery in plants. Nature Protocols (2020). DOI: 10.1038/s41596-020-0370-0 Demirer, G.S., Zhang, H., Goh, N.S., Chang, R., Landry, M.P.‡ Nanotubes effectively deliver siRNA to intact plant cells and protect siRNA against nuclease degradation. Science Advances (2020). 6 DOI: 10.1126/sciadv.aaz0495 Cunningham, F.J., Demirer, G.S., Goh, N.S., Zhang, H., Landry, M.P.‡. Nanobiolistics: An Emerging Genetic Transformation Approach. Biolistic DNA Delivery in Plants (2020). pp 141-159 Additionally, we have presented our work at the following conferences for which we were invited to give talks relevant to efforts of this grant: University of Minnesota NIH Chemistry-Biology Interface Research Symposium. (Invited): Nanomaterials Engineering for Biomolecule Delivery to Plants. Minneapolis, MN. (May 2021) ECS 239th Meeting - 2021 - 2020 Nanocarbons Division SES Young Investigator Award (Invited): Nanomaterials Engineering to Probe and Control living Systems. Chicago, IL. (May 2021) UCLA Department of Chemistry and Biochemistry Seminar (Invited): Tiny things to engineer solutions for life's big problems. Los Angeles, CA. (May 2021) 3M Seminar Series (Invited): Nanomaterials Engineering to Probe and Control Living Systems. St. Paul, MN. (April 2021) University of Florida Department of Chemical Engineering Seminar (Invited): Nanomaterials to probe and control biological systems.. Gainesville, FL. (April 2021) University of Cincinnati Chemistry (Invited): Tiny things to engineer solutions for life's big problems. Cincinnati, OH. (April 2021) Rowan University Physics Department Seminar (Invited): Tiny things to engineer solutions for life's big problems. Glassboro, NJ. (March 2021) Carnegie Mellon University Department of Materials Science and Engineering Seminar (Invited): Nanomaterials Engineering to Probe and Control Living Systems. Pittsburgh, PA. (February 2021) Harvard University School of Engineering and Applied Sciences Seminar (Invited): Nanomaterials Engineering to Probe and Control Living Systems. Pittsburgh, PA. (January 2021) University of California Riverside Plant Sciences Seminar (Invited): Nanomaterials enable delivery of genetic material to plants without transgene integration. Riverside, CA. (January 2021) Max Planck Institute of Biochemistry (Invited): Nanomaterials enable delivery of genetic material to plants without transgene integration. Munich, Germany. (January 2021) Corteva Plant Breeding, Genetics, and Biotechnology (PBGB) symposium, Michigan State University (Invited): Nanomaterials enable delivery of genetic material to plants without transgene integration. East Lancing, MI. (December 2020) University of California Berkeley Department of Molecular and Cellular Biology Seminar (Invited): Nanomaterials Engineering to Probe and Control Living Systems. Berkeley, CA. (December 2020) Pacifichem 2020 (Invited): Carbon Nanotubes Enable Delivery of Genetic Material Without Transgene Integration in Mature Plants. Honolulu, HI. (December 2020) *Postponed due to covid-19 Plant Sciences Institute, Israeli National Center for Genome Editing in Agriculture (Invited): Nanomaterials enable delivery of genetic material to plants without transgene integration. Rishon, Israel (November 2020) Naval Research Laboratory Center for Bio/Molecular Science and Engineering (Invited): Nanomaterials enable delivery of genetic material to plants without transgene integration. (November 2020) Sustainable Nanotechnology Organization Meeting (Invited): Nanomaterials enable delivery of genetic material to plants without transgene integration. Virtual meeting. (November 2020) ACS Nano Rising Star Symposium (Invited): Engineering Nanomaterials for Biotechnology. Beijing, China. (November 2020) NANO FOR AGRI 2020: Application of Nanotechnology for Sustainable, Productive and Safer Agriculture and Food Systems, TERI-Deakin Nanobiotechnology Centre 2020 (Invited): Nanomaterials Enable Delivery of Genetic Material Without Transgene Integration in Mature Plants. Gurugram, India. (November 2020) AIChE Annual Meeting, Bionanotechnology Division (Invited Plenary): Nanomaterials Engineering to Probe and Control Living Systems. San Francisco, CA. (November 2020) Carnegie Mellon University Department of Chemical Engineering Seminar (Invited): Nanomaterials Engineering to Probe and Control Living Systems. Pittsburgh, PA. (November 2020) UNC Eshelman School of Pharmacy - Rising Stars in Drug Delivery and Novel Carriers Seminar (Invited): Nanomaterials enable delivery of genetic material to plants without transgene integration. Chapel Hill, NC. (November 2020) Rice University Department of Bioengineering Seminar (Invited): Nanomaterials Engineering to Probe and Control Living Systems. Houston, TX (October 2020) Plant Genome Editing Symposium - Society for the Advancement of Chicanos and Native Americans In Science, SACNAS (Invited): Nanomaterials enable delivery of genetic material to plants without transgene integration. Long Beach, CA. (October 2020) 4th International Conference on Plant Synthetic Biology, Bioengineering, and Biotechnology (Invited): Nanomaterials enable delivery of genetic material to plants without transgene integration. College Copenhagen, Denmark. (October 2020) Texas A&M Genome Editing Symposium (Invited Keynote): Nanomaterials enable delivery of genetic material to plants without transgene integration. College Station, TX. (October 2020) 8th Plant Genomics & Gene Editing Congress (Invited Keynote): Nanomaterials enable delivery of genetic material to plants without transgene integration. Raleigh, NC. (October 2020) *Postponed due to covid-19 Department Biomedical Engineering at City College New York Seminar (Invited): Nanotechnologies for life's big problems. New York, NY. (September 2020) ETH Zurich Department of Biology (Invited): Nanomaterials enable delivery of genetic material to plants without transgene integration. Zurich, Switzerland. (September 2020) SciX 2020 Conference (Invited Keynote): Emerging Leader in Molecular Spectrocopy: Nanomaterials Engineering for Life Science Applications. Sparks, NV. (September 2020) ? What do you plan to do during the next reporting period to accomplish the goals?We are glad to report successful completion of most of our project goals in publication or patent form, except for our ongoing project involving the delivery of RNPs to plants with nanomaterials. The reason for this delay is that, due to COVID-19, we lost access to our research space starting March 2020 - July 2020. As of July 2020, we are operating under ~15% capacity, which has significantly limited our ability to carry out our genome editing studies. We also lost our transgenic plant lines, and are currently working on acquiring new seeds and planting the transgenic Cas9-Nb and mGFP5 lines needed for our project. Lastly, we have recently been able to regain access to our greenhouse facilities, and are back to full-scale research operation. The Landry lab reopened at 10% capacity in July 2020 and more recently 50% capacity in April 2021 and will next work on validating the preliminary constructs generated with carbon nanotube-based suspension of sgRNA, mRNA, and donor DNA templates. Specifically, we have re-generated our Cas9-expressing Nb line and will test the ability of the aforementioned constructs to enable or increase the efficiency of CRISPR-based genome editing. Our next steps will be to perform our RNP delivery tests in leaves first, before testing nanoparticle internalization in tissue culture preparations for future testing of editing efficiencies in regenerable tissues.
Impacts
What was accomplished under these goals?
We have developed a ratiometric dual-color luciferase reporter assay to quantify transcriptional activity of genetic elements in plants. This reporter system is based on two luciferases that process the same substrate and emit light at different wavelengths. This system is i) ratiometric, with both reporters measured by the same method and at the same time, ii) has a high dynamic range output that allows precise comparison of a wide range of expression magnitudes, iii) requires minimal sample preparation time and, iv) is cheaper than currently used dual luciferase methods. This reporter assay will allow us to expedite nanoparticle-based testing of reporter expression. To this end, we surveyed a panel of luciferases that emit green or red luminescence by transiently expressing them in Nicotiana benthamiana leaves by agroinfiltration. We extracted total protein from leaf tissues and measured luciferase emission spectra and stability. We selected E-Luc and Red-F as green and red emitting luciferases, respectively, because this pair of luciferases exhibited the most distant emission peaks. Additionally, both luciferases showed very similar and stable reaction kinetics that allow reliable relative measurements starting 10 minutes after extract and substrate mixture, with stable luminescence for over 2 hours. These properties allow characterization of a large number of samples in a standard luminometer without the need for expensive injectors required for time-sensitive coelenterazine-using luciferases. Moreover, we were able to measure stable luminescence directly from leaf discs. This method greatly reduces sample processing time, and thus, is amenable for high throughput measurements. Additionally, we developed the use of gold nanoparticles of various shapes and sizes to uncover the transport mechanism of nanoparticles in plant tissues, and also study the availability of biological cargoes as they are transported within plant tissues. Our study uncovers that nanoparticle shape and size both greatly influence nanoparticle uptake and the ability of plants to utilize the delivered biological cargoes, with a strict 20 nm size cut-off. Specifically, we attach small interfering RNA (siRNA, a notoriously fragile cargo), and assess the mechanism of the plant's ability to use siRNA for targeted downregulation of a reporter green fluorescent protein (GFP). We uncover several surprising and counter-intuitive findings as part of our study, specifically: Gold nanospheres (0D nanomaterials) do not internalize into plant cells - regardless of how small they are - whereas gold nanorods (1D nanomaterials) do internalize. It takes on the order of hours (quite long!) for nanoparticles to reach plant cells. Counter to expectation, these results mean that simple diffusion is not the main mechanism for nanoparticle transport in plants. Nanoparticle transport into plant cells is an energy-dependent endocytosis-driven process. Smaller nanoparticles, and rod-shaped nanoparticles, reach plant cells faster than larger or spherical nanoparticles. Despite the fact that gold nanospheres do not enter plant cells, 10 nm gold nanospheres are most effective at delivering siRNA cargoes into cells. This finding is particularly important for the plant nano-delivery community, as it suggests that nanoparticle entry into cells is not necessary for siRNA cargo delivery. We highlight this latter finding as a potential 'paradigm shifter' for the field of nanoparticle-based delivery in plants. There is currently much effort in confirming internalization of nanoparticles into plant cells prior to their use as delivery vehicles. Our results highlight that the best delivery nanotechnologies may not need to enter plant cells at all. Our manuscript therefore describes mechanisms of nanoparticle transport in plants to motivate how future endeavors plant delivery should be approached. Lastly, the example application in our work demonstrates targeted downregulation of plant genes by delivering siRNA with gold nanoparticles (and confirming the counterintuitive nanoparticle shape- and size- dependent performance). The biocompatibility of gold nanoparticles makes our technology an attractive "green" alternative to the use of pesticides in agriculture via downregulation of a specific plant protein via sequence-specific destruction of the plant protein's messenger RNA. To our knowledge, this work represents the first-time usage of gold nanoparticles in plants, without biolistics, for nucleic acid delivery. To our knowledge, this is also the first systematic assessment of how nanoparticle shape and aspect ratio affects nanoparticle transport at the cellular level.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Heller, D.!, Jena, P., Pasquali, M. Kostarelos, K.,& Landry, M.P., Wenseleers, W., Yudaska, M. Banning carbon nanotubes would be scientifically unjustified and damaging to innovation. Nature Nanotechnology (2020). 15, 164166
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Demirer, G.S., Zhang, H., Goh, N.S., Chang, R., Landry, M.P.! Carbon nanocarriers deliver siRNA to intact plant cells for efficient gene knockdown. Science Advances (2020). 6 (26).
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zhang, H., Zhang, H., Demirer, G.S., Gonzales-Grandio, E., Fan, C., Landry, M.P.! Engineering DNA nanostructures for siRNA delivery in plants. Nature Protocols (2020). 15, 30643087
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Hofmann, T., Lowry!, G.V.!, Ghoshal, S., Tufenkji, N., Brambilla, D., Dutcher, J.R., Gilbertson, L.M., Giraldo, J. P., Kinsella, J. M., Landry, M.P., Lovell, W., Naccache, R., Paret, M., Pedersen, J. A., Unrine, J., M., White, J.C., Wilkinson, K.J. Technology readiness and overcoming barriers to sustainably implement nanotechnology-enabled plant agriculture. Nature Food (2020). 1, 416425
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Demirer, G.S.!, Landry, M.P.! Efficient Transient Gene Knock-down in Tobacco Plants Using Carbon Nanocarriers. Bio-protocol (2021). 11(1)
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Demirer, G.S.!, Silva, T.N., Jackson, C.T., Thomas, J.B., Ehrhardt, D., Rhee, S.Y.!, Mortimer, J.C.!, Landry, M.P.! Nanotechnology to advance CRISPR/Cas genetic engineering of plants. Nature Nanotechnology (2021). 16, 243250
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Wang, J., Cunningham, F. J., Goh, N., Boozarpour, N.N., Pham, M., Landry, M.P.! A nanoparticle approach for protein delivery in planta. Current Opinion in Plant Biology (2021). 2124, 141-159
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Voke, E., Pinals, R.L., Goh, N., Landry, M.P.! In Planta Nanosensors: Understanding Bio-corona Formation for Functional Design. ACS Sensors (2021)
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Wang, J.W., Cunningham, F.J., Goh, N., Boozarpour, N.N., Pham, M., Landry, M.P.! Nanoparticles for protein delivery in planta. Current Opinion in Plant Biology (2021). 60 (102052).
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Zhang, H., Cao, Y., Xu, D., Goh, N.S., Demirer, G.S., Chen, Y., Landry, M.P.!, Yang, P.! Gold nanocluster mediated delivery of siRNA to intact plant cells for efficient gene knockdown. Nano Letters (2021)
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Zhang, H.*, Goh, N.S.*, Wang, J., Demirer, G.S., Butrus, S., Park, S-J, Landry, M.P.! Nanoparticle Cellular Internalization is Not Required for RNA Delivery to Mature Plant Leaves. Nature Nanotechnology (2021)
" Highlighted in Nature Materials (2021), C. Horejs
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Grandio-Gonzalez, E., Demirer, G.S., Ma, W., Brady, S.M., Landry, M.P.! A ratiometric dual color luciferase reporter for fast characterization of transcriptional regulatory elements. ACS Synthetic Biology (2021)
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Gonzalez-Grandio, E., Demirer, G.S., Jackson, C.T., Yang, D., Landry, M.P.! Carbon nanotube biocompatibility in plants is determined by their surface chemistry. Journal of Nanobiotechnology (2022)
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Progress 04/01/20 to 03/31/21
Outputs
Target Audience:The target audience are researchers in plant biology and biotechnology, and synthetic biology Changes/Problems:COVID-19 has greatly slowed our research progress for the reporting period since we are operating at 15% lab capacity. We have limited access to shared facilities needed for nanoparticle synthesis, characterization, and plant growth. We hope to resume regular activities in late 2021. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?We have disseminated our results in publications, and in numerous talks at conferences and seminars. What do you plan to do during the next reporting period to accomplish the goals?Previously, observations of toxicity arising from PEI-CNT application to mature leaves was qualitative and associated with visible necrosis. To quantitatively gauge and better understand the effect of PEI-CNT on plant leaves, toxicity marker genes associated with PEI-CNT exposure had to be identified. To this end, RNAseq was carried out with Arabidopsis thaliana. We showed differentially expressed gene clusters across three conditions - PEI-CNTs, pristine CNTs loaded with ssRNA, and water with five biological replicates. Using this data set, a set of genes that were maximally upregulated and downregulated with PEI-SWNTs compared to the water control was selected. The corresponding orthologs in Nicotiana benthamiana were identified, and a RT-qPCR analysis was conducted. PR1, the most upregulated gene, is a pathogenesis related protein and has been used as a marker for biotic stress. AT3G54830, amongst the genes that are most downregulated, is a putative transmembrane amino acid transporter family protein of unknown function. Across the board with Arabidopsis thaliana, both gene expression changes with PR1 and AT3G54830 demonstrate a logical trend with increasing PEI-SWNT concentrations. These respective upward and downward trends are also conserved with the relevant Nb orthologs. Of note, the inclusion of carboxylated CNT samples induced no significant gene expression level change in either gene, suggesting that the response may be due to the presence of the cationic polymer, PEI. To this end, we have been testing alternate polymers that are more biocompatible than PEI. We will create a colloidally-stable polymer-SWNT construct capable of loading high amounts of pDNA. This involves adapting covalent functionalization efforts in published papers, followed by efforts to increase SWNT solubility and analyze the extent of pDNA loading on these constructs. Promising chemistries that have been identified include pyrrolidine (Paiva et al., 2010), triazine (Setaro et al., 2017), and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (Baker et al., 2002; Liu et al., 2009) functionalization of SWNTs. Initial work has been done with pyrrolidine and triazine functionalization, with the former put on hold due to low efficiency of functionalization and low solubility in aqueous solutions. More extensive experiments have been conducted with triazine chemistry, which enables SWNT surface functionalization whilst preserving π-network on SWNTs (Chio et al., 2020). The triazine compound further allows the addition of an amine-containing compound, which is amenable to polymer attachment to SWNTs. Several small molecules and polymers containing free amines have been conjugated to triazine-SWNTs, and their zeta potentials measured. Preliminarily, these zeta potentials suggest polymer-based conjugates display larger zeta potential magnitudes and thus higher colloidal stability. Triazine-SWNT conjugation with polymers has been conducted with several biocompatible cationic polymers. Additionally, there are many directions towards further developing this technology, and next steps for this work will continue to focus on troubleshooting the existing polymer-CNT chemistries and systems within the Landry Lab, as well as furthering Aim 2, which can currently proceed without the existence of functional polymer-CNT constructs. Alternatives to pDNA delivery - such as siRNA or mRNA delivery - may be a future direction forour project. Specific goals and pursuits are as follows: Aim 1 Testing the expression using DNA-polymer-CNTs in various plant systems remains contingent on the Landry Lab being able to produce functional polymer-CNT constructs. Similarly, testing DNA-polymer-CNTs in rice calli within the Landry Lab has also be put on hold. Aim 2 The Landry Lab will continue to study the relative mRNA levels of NbPR1a in response to the list of biocompatible polymers by conducting RT-qPCR experiments with a larger quantity of biological replicates. Using the toxicity responses from the 25k bPEI (original polymer used in PEI-CNTs) as a benchmark, 4-8 polymers will be identified as inducing a lower degree of toxic response. Following from preliminary tests, PR1 upregulation is likely due to the presence of the cationic polymer and not the CNT. Nevertheless, the toxic response will be verified with polymer suspended CNTs to substantiate PR1 expression results with the free polymer. The shortlisted group of polymers will then be utilized in downstream experiments for polymer-CNT conjugation and expression validation. Optimization of polymer-CNT chemistry - Troubleshooting PEI-CNT synthesis conditions Significant time and effort have been put into troubleshooting different aspects of PEI-CNT synthesis, including reagent ratios, starting material purity, type of conjugation reaction, and pH of wash solutions. None of these modified parameters induced a notable change in final construct zeta potential nor improvement in pDNA delivery and expression. This approach will not be further pursued as it will be more valuable to pursue other more promising avenues. Optimization of polymer-CNT chemistry - Development of polymer-CNT conjugation chemistries Synthesis of covalently modified CNTs is promising, and work will continue in this area. The Landry Lab will continue to investigate the feasibility of triazine-CNT chemical modifications as a method to generate covalently grafted polymer-CNTs in four distinct steps. This first will include testing biocompatible cationicpolymers with available amines for triazine-CNT grafting, quantifying suspension yield and zeta potential. Second, if constructs experience low water solubility, a library of amphiphilic molecules can be utilized to increase hydrophilicity. Third, following the generation of polymer-triazine-CNTs that are soluble and colloidally stable in water, the pDNA loading capacity of the polymer-CNTs will be quantified via running DNA-polymer-CNTs on agarose gels to verify pDNA retardation. Lastly, DNA-polymer-CNTs will be infiltrated into Nb leaves and tested for luciferase expression 3 days post-infiltration.
Impacts
What was accomplished under these goals?
To reliably test the efficacy of PEI-SWNT constructs between synthesis batches as well as compare efficacy polymer-SWNTs between different polymers, we needed a robust testing system that ideally provided a quantitative measure of delivery efficiency. As the end goal of this platform is to establish a delivery method that will result in high levels of genome editing machinery in a plant cell nucleus, the most direct measure of pDNA delivery efficiency would entail measuring expression levels of the gene encoded by the pDNA. Initially, the lab exclusively utilized GFP as a fluorescent reporter paired with confocal microscopy for visible expression validation. However, this method poses several challenges, including that detection of low levels of GFP expression necessitate high laser power or digital gain, both of which increase background signal from auto fluorescent compounds present and make it difficult to ascertain if expression has occurred. Furthermore, GFP expression by PEI-SWNTs is often not uniform across tissue and requires rastering across the entire sample, making this method not amenable to high-throughput screening of polymer-SWNT constructs. Efforts to address these challenges were two-fold. First, the lab created new plasmid constructs encoding for GFP containing a nuclear localization signal (NLS GFP). As opposed to the initial GFP pDNA that nonspecifically localized within the cytosol that can mimic signal from autofluorescence or tissue damage, expression of the NLS GFP pDNA would provide higher signal confidence with the observation of distinct puncta corresponding to the cell nucleus. UsingAgrobacteriumfor pDNA delivery, the lab demonstrated that the use of NLS GFP pDNA permits imaging at lower digital gain and laser power, reducing background signal. Second, a transition towards quantitative assays via plate readers would enable screening throughput of infiltrated samples, combined with the use of a luminescence-based reporter such as luciferase. Several luciferase proteins from different organisms (firefly, Renilla, click beetle) and reporter assay conditions were tested to obtain an optimal signal/background ratio with the least sample processing, favoring the ability to perform high-throughput analyses. We demonstrated high levels of detectable fluorescence and luminescence with GFP and luciferase-encoding pDNA constructs expressed inN. benthamianaleaves viaAgrobacterium-mediated delivery. While high signal level was achieved with both GFP and luciferase, the nature of luciferase as a chemiluminescent assay enables lower background as exemplified by the lower signal obtained with negative controls. Going forward, the lab has been primarily probing and quantifying PEI-CNT delivery and downstream expression with the luciferase plate assay. The Landry lab has also procured rice calli as a secondary system for PEI-CNT validation. The use of rice calli is desirable as a form of well-documented immature plant tissue. Additionally, growth conditions of rice calli can be tuned to influence the thickness of the cell wall and cell-to-cell access (i.e. friable vs compact), providing a system with tunable knobs for future study with PEI-CNTs. As PEI-CNT synthesis occurs in a non-sterile environment, the lab has developed a protocol for the sterilization of PEI-CNTs. This involves overnight exposure of the PEI-CNT solution to a UV light source, and confirmed UV-treated samples does not experience visible contaminants. Out of three replicates, one bacterial colony was observed with a UV-treated PEI-CNT sample after 5 days. It is likely that either increased UV exposure time or the use of a tube rotator will enhance the effectiveness of this sterilization protocol. Microwave-sterilization was also considered, but progress moved in favor of UV-sterilization due to the relative ease and amenability to high-throughput sterilization methods. The rice calli system has also been successfully validated once with luciferase expression with the use ofAgrobacterium. Initial testing delivering luciferase-encoding pDNA with PEI-CNTs in rice calli did not result in any detectable levels of expression, though no conditions have yet been optimized. Lastly, we tested editing efficiency via delivery of a gRNA plasmid to mutantNicotiana benthamiana (Nb)plant that expresses Cas9 protein constitutively. We delivered plasmids containing a gRNA cassette targeting knock-out of Phytoene desaturase (PDS) in intactNbleaves with PEI-CNTs orAgrobacterium. This gRNA targets a MlyI restriction enzyme site on the PDS gene, so that the editing rate can be quantified with restriction site mutation assay (RE), T7 Endonuclease I (T7E1) assay, and indel tracking with Sanger sequencing (TIDE). The plasmids are loaded on PEI-CNT vehicles and introduced into intact leaves via infiltration, with delivery of the same plasmid withAgrobacteriumas a positive control. Infiltrated plants undergo a heat treatment (5 cycles of 22?C 24 h and 37?C 24 h) to improve Cas9 activity. 10-days post-infiltration, non-treated, PEI-CNTs, andAgro-infiltrated leaves are collected for genomic DNA extraction and amplification of the PDS gene for editing quantification by RE, T7E1, and TIDE. These preliminary results show at 15% and 35% editing rate in PEI-CNT andAgrobacteriumtreated samples, respectively.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2021
Citation:
Demirer, G.S.!, Silva, T.N., Jackson, C.T., Thomas, J.B., Ehrhardt, D., Rhee, S.Y.!, Mortimer, J.C.!, Landry, M.P.! Nanotechnology to advance CRISPR/Cas genetic engineering of plants. Nature Nanotechnology (2021).
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Hofmann, T., Lowry!, G.V.!, Ghoshal, S., Tufenkji, N., Brambilla, D., Dutcher, J.R., Gilbertson, L.M., Giraldo, J. P., Kinsella, J. M., Landry, M.P., Lovell, W., Naccache, R., Paret, M., Pedersen, J. A., Unrine, J., M., White, J.C., Wilkinson, K.J. Technology readiness and overcoming barriers to sustainably implement nanotechnology-enabled plant agriculture. Nature Food (2020). DOI: 10.1038/s43016-020-0110-1
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Demirer, G.S., Zhang, H., Goh, N.S., Chang, R., Landry, M.P.! Nanotubes effectively deliver siRNA to intact plant cells and protect siRNA against nuclease degradation. Science Advances (2020). 6 DOI: 10.1126/sciadv.aaz0495
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Demirer, G.S., Zhang, H., Goh, N.S., Grandio, E.G., Landry, M.P.! Carbon nanotube-mediated DNA delivery without transgene integration in intact plants. Nature Protocols, (2019). p 29542971
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Landry, M.P.!, Mitter, N.! How nanocarriers delivering cargoes in plants can change the GMO landscape. Nature Nanotechnology (2019). 14; pp. 512514
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zhang, H., Zhang, H., Demirer, G.S., Gonzales-Grandio, E., Fan, C., Landry, M.P.! Engineering DNA nanostructures for siRNA delivery in plants. Nature Protocols (2020). DOI: 10.1038/s41596-020-0370-0
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Progress 04/01/19 to 03/31/20
Outputs
Target Audience:The target audience is the plant biotechnology research community for this reporting period. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?I have mentored numerous trainees in my lab: 13 undergraduate researchers, 17 graduate students, and 8 postdoctoral trainees. I have graduated one student who is now a group leader at HHMI's Janelia Research Campus. The student who is leading the nanoparticle-based gene delivery project, Gozde Demirer, has interviewed for faculty positions and has multiple offers at R1 research universities. Additionally, 6 students and postdocs have presented their research in this space at conferences in the past year. How have the results been disseminated to communities of interest?We have published a series of peer reviewed publications to disseminate our results. Additionally, we have presented our work at the following conferences for which we were invited to give talks: Society for In Vitro Biology (Invited Keynote): High Aspect Ratio Nanomaterials Enable Delivery of Functional Genetic Material Without DNA Integration in Mature Plants. Tampa, FL (June 2019) Umeå Plant Science Centre (Invited): High Aspect Ratio Nanomaterials Enable Delivery of Functional Genetic Material Without DNA Integration in Mature Plants. Umeå, Sweden (May 2019) 2019 Yale Chemical Biology Symposium (Invited): Nanomaterials Engineering to Probe and Control Living Systems. New Haven, CT (May 2019) UC Berkeley Department of Bioengineering (Invited): Nanomaterials Engineering to Probe and Control Living Systems. Berkeley, CA (May 2019) AIChE Midwest Meeting (Invited Keynote): Nanomaterials Engineering to Probe and Control Living Systems. Chicago, IL (March 2019) Rockefeller University Center for Studies in Physics and Biology (Invited): Nanomaterials Engineering to Probe and Control Living Systems. New York, NY (February 2019) Memorial Sloan Kettering Cancer Center of Molecular Imaging and Nanotechnology (Invited): Nanomaterials Engineering to Probe and Control Living Systems. New York, NY (February 2019) UCSD Plant Biology Symposium (Invited): Genetic Transformation of Plants with Nanoparticles. San Diego, CA (January 2018) KAUST Plant Sciences Program (Invited): High Aspect Ratio Nanomaterials Enable Transgene Expression and Silencing in Plants. Thuwal, Saudi Arabia (September 2018) American Chemical Society Applied Nanotechnology for Food & Agriculture: High Aspect Ratio Nanomaterials Enable Biomolecule Delivery and Transgene Expression or Silencing in Mature Plants. Boston, MA (August 2018) What do you plan to do during the next reporting period to accomplish the goals?The importance of our work lies in the fact that plant and crop genetic engineering can solidify the agricultural industry by conferring desirable traits to plants such as increased yield, abiotic stress tolerance, and disease and pest resistance. Current biomolecule delivery methods to plants experience limitations that hinder their delivery efficacy and are largely ineffective in consumption crops. My lab's nanoparticle-based platform enables delivery of gene vectors to plants, inclusive of crop plant species, such as wheat and cotton. Broad-scale production of transgenic plants, enabled by this high-throughput and high-yield transformation platform, can address the need for sustainable and high-yielding crops. Furthermore, our platform for delivery of Cas9 DNA vectors represents a platform by which to genetically modify plants without DNA integration (when delivering a DNA vector coding for CRISPR), or without DNA altogether (when delivering an RNP), enabling these gene-edited crops to circumvent the costly process and negative public perception of GMO labeling. Motivated by this, we will next implement carbon nanotube mediated plasmid DNA delivery for the delivery and transient expression of CRISPR plasmids, to enable permanent edits to the plant genome without transgenic DNA integration. Specifically, we will codon and promoter optimize CRISPR plasmids for delivery to monocot and dicot plants. We will implement our nanotechnology for genome editing in Nb via delivery of DNA CRISPR-Cas9 vectors or RNP genome editing machinery to demonstrate plant genome editing without transgenic DNA integration. We will also optimize transformations in tissue culture preparations (embryonic tissue, callus, germ line cells, seeds, pollen) to test editing efficiencies in regenerable tissues. Following DNA plasmid construct optimization, CRISPR editing efficiency testing with both nuclear and chloroplast genome editing will be systematically assessed in both leaves and callus. Excitingly, preliminary results show between 9 and 30% genome editing efficiency of CRISPR plasmids delivered to Nb leaves following a single infiltration of nanotubes loaded with a non-optimized CRISPR plasmid.
Impacts
What was accomplished under these goals?
This project is involves a systematic study of nanoparticle size, shape, aspect ratio, charge, and tensile strength for DNA, RNA, and protein delivery towards plant genome editing. Success in nanoparticle-mediate delivery of gene editing tools will produce a platform by which to genetically modify plants without DNA, which could transform our view of genetically modified foods. The present study is founded at the interface of nanomaterial science and plant biotechnology, will systematically unravel the complexity of nanomaterial interactions and transport in plants for gene editing. We recently demonstrated the generation and application of various nanomaterials and their associated surface chemistries to controllably graft and quickly deliver whole DNA plasmids and siRNA to various agriculturally-relevant plants (Demirer et al. Nature Nanotechnology 2019; Zhang et al. PNAS 2019; Demirer et al. Sci Adv, in press, 2020). With carbon nanotube mediated DNA delivery, we accomplished nanoparticle-mediated heterologous expression of a protein in plants with strong and transient expression, and show that there is no transgenic DNA integration into the host plant genome. We demonstrated both gene expression and gene silencing in both model and non-model plant species of agricultural relevance: Nicotiana benthamiana (tobacco, model dicot plant), Eruca sativa (arugula, non-model dicot plant); Triticum aestivum (wheat, non-model monocot plant) and Gossypium hirsutum (cotton, non-model dicot plant). Wheat and cotton had been suggested by crop scientists, and in particular cotton which is very challenging to genetically transform. We further demonstrated with single-molecule imaging that the process of grafting functional RNA on these nanomaterials actually prevents DNA degradation by nucleases, providing a complete picture of how this nanotechnology both promotes delivery of functional biomolecules to plants, and also protects the cargo from enzymatic degradation en route to its intracellular function. UC Berkeley has filed an international patent on these results, which have drawn licensing interest from three companies in the plant biotechnology space (Bayer/Monsanto, Syngenta, BASF). Since we began presenting this work, we have shipped dozens of samples to laboratories and agricultural companies domestically and internationally, which have shown transformation success in their hands for even more crop species and applications.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Wang, J.W, Grandio, E.G., Newkirk, G.M., Demirer, G.S., Butrus, S., Giraldo, J.P.!, Landry, M.P.! Nanoparticle-mediated genetic engineering of plants. Molecular Plant (2019) DOI: 10.1016/j.molp.2019.06.010
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Zhang, H.*, Demirer, G.S.*, Zhang, H., Ye, T., Goh, N.S., Aditham, A.J., Cunningham, F.J., Fan, C., Landry, M.P.! Low-dimensional DNA Nanostructures Coordinate Gene Silencing in Mature Plants. PNAS (2019). DOI: 10.1073/pnas.1818290116
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Chio,L., ODonnell, J.T., Kline, M., Kim, J.H., McFarlane, I.R., Zuckermann, R.N., Landry, M.P.! Electrostatic-assemblies of single-walled carbon nanotubes and sequence-tunable peptoid polymers detect a lectin protein and its target sugars. Nano Letters (2019). DOI: 10.1021/acs.nanolett.8b04955
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Demirer, G.S., Zhang, H., Goh, N.S., Grandio, E.G., Landry, M.P.! Carbon nanotube-mediated DNA delivery without transgene integration in intact plants. Nature Protocols (2019). DOI: 0.1038/s41596-019-0208-9
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Landry, M.P.!, Mitter, N.! How nanocarriers delivering cargoes in plants can change the GMO landscape. Nature Nanotechnology 2019, 14; pp. 512514
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Pinals, R.L.*, Yang, D.*, Lui, A., Cao, W., Landry, M.P.! Corona exchange dynamics on carbon nanotubes by multiplexed fluorescence monitoring. JACS (2019) DOI: 10.1021/jacs.9b09617
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Chio, L., Pinals, R., Murali, A., Goh, N.S., Landry, M.P.! Surface Modification Effects on Single-Walled Carbon Nanotubes for Multimodal Optical Applications. Advanced Functional Materials (2020). DOI: 10.1002/adfm.201910556
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