Carbon nanotube–mediated DNA delivery without transgene integration in intact plants

Abstract

Exogenous biomolecule delivery into plants is difficult because the plant cell wall poses a dominant transport barrier, thereby limiting the efficiency of plant genetic engineering. Traditional DNA delivery methods for plants suffer from host-species limitations, low transformation efficiencies, tissue damage, or unavoidable and uncontrolled DNA integration into the host genome. We have demonstrated efficient plasmid DNA delivery into intact plants of several species with functionalized high-aspect-ratio carbon nanotube (CNT) nanoparticles (NPs), enabling efficient DNA delivery into a variety of non-model plant species (arugula, wheat, and cotton) and resulting in high protein expression levels without transgene integration. Herein, we provide a protocol that can be implemented by plant biologists and adapted to produce functionalized single-walled CNTs (SWNTs) with surface chemistries optimized for delivery of plasmid DNA in a plant species–independent manner. This protocol describes how to prepare, construct, and optimize polyethylenimine (PEI)-functionalized SWNTs and perform plasmid DNA loading. The authors also provide guidance on material characterization, gene expression evaluation, and storage conditions. The entire protocol, from the covalent functionalization of SWNTs to expression quantification, can be completed in 5 d.

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Fig. 1: Overview of the procedure for plant transformation with PEI-SWNTs.
Fig. 2: Probe-tip sonication setup.
Fig. 3: Suggested usage of Amicon 100,000-MWCO filters for washing.
Fig. 4: Stability of the DNA-loaded PEI-SWNT suspension.
Fig. 5: Infiltration of leaves with DNA-loaded PEI-SWNTs.
Fig. 6: Anticipated results.

Data availability

All materials are available from commercial sources or can be derived using methods described in this study. All primary data underlying the figures reported in the article can be obtained from the corresponding author upon reasonable request.

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Acknowledgements

We acknowledge support from a Burroughs Wellcome Fund Career Award at the Scientific Interface (CASI), a Stanley Fahn PDF Junior Faculty Grant under award no. PF-JFA-1760, a Beckman Foundation Young Investigator Award, a USDA AFRI award, a grant from the Gordon and Betty Moore Foundation, a USDA NIFA award, a USDA-BBT EAGER award, support from the Chan-Zuckerberg Foundation, and an FFAR New Innovator Award (to M.P.L.). G.S.D. was supported by a Schlumberger Foundation Faculty for the Future Fellowship. H.Z. acknowledges the support of the National Natural Science Foundation of China (21605153). We also acknowledge support from the UC Berkeley Molecular Imaging Center (supported by the Gordon and Betty Moore Foundation), the QB3 Shared Stem Cell Facility, and the Innovative Genomics Institute (IGI). We are grateful to the Staskawicz lab at UC Berkeley for the N. benthamiana seeds.

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Contributions

G.S.D. and M.P.L. designed the experiments. G.S.D., H.Z., and N.S.G. performed the experiments. G.S.D. analyzed the data and created the figures. G.S.D., H.Z., N.S.G., and E.G.G. wrote the manuscript. All authors approved the manuscript.

Corresponding author

Correspondence to Markita P. Landry.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Ardemis Boghossian, Mohammad Ramezani and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Demirer, G. S. et al. Nat. Nanotechnol. 14, 456–464 (2019): https://www.nature.com/articles/s41565-019-0382-5

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Supplementary Figure 1 Characterization and comparison of heat reaction and EDC/NHS coupling for the synthesis of PEI-SWNTs.

a, XPS nitrogen peaks for the heat reacted PEI-SWNTs: amine peak at 399 eV and amide peak at 401 eV. b, XPS nitrogen peaks for the EDC/NHS reacted PEI-SWNTs. c, Zeta potential measurements of COOH-SWNTs, PEI-SWNTs via heat reaction, and DNA loaded PEI-SWNTs. ****P < 0.0001 and *P = 0.0191 in two-way ANOVA. N=3 and error bars are standard deviation. d, Zeta potential measurements of COOH-SWNTs, PEI-SWNTs via EDC/NHS coupling, and DNA loaded PEI-SWNTs. ****P < 0.0001and **P = 0.0025 in two-way ANOVA. N=3 and error bars are standard deviation. e, Representative confocal images at Day 3 and 10 of wild-type Nicotiana benthamiana leaves infiltrated with DNA-PEI-SWNTs prepared via heat reaction. e, Representative confocal images at Day 3 and 10 of wild-type Nicotiana benthamiana leaves infiltrated with DNA-PEI-SWNTs prepared via EDC/NHS coupling. All scale bars are 50 µm.

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Demirer, G.S., Zhang, H., Goh, N.S. et al. Carbon nanotube–mediated DNA delivery without transgene integration in intact plants. Nat Protoc 14, 2954–2971 (2019). https://doi.org/10.1038/s41596-019-0208-9

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