Gold-Nanocluster-Mediated Delivery of siRNA to Intact Plant Cells for Efficient Gene Knockdown

Creators: Zhang, Huan; Cao, Yuhong; Xu, Dawei; Goh, Natalie S.; Demirer, Gozde S.; Cestellos-Blanco, Stefano; Cheng, Yuan; Landry, Markita P.; Yang, Peidong

Abstract

RNA interference, which involves the delivery of small interfering RNA (siRNA), has been used to validate target genes, to understand and control cellular metabolic pathways, and to use as a "green" alternative to confer pest tolerance in crops. Conventional siRNA delivery methods such as viruses and Agrobacterium-mediated delivery exhibit plant species range limitations and uncontrolled DNA integration into the plant genome. Here, we synthesize polyethylenimine-functionalized gold nanoclusters (PEI-AuNCs) to mediate siRNA delivery into intact plants and show that these nanoclusters enable efficient gene knockdown. We further demonstrate that PEI-AuNCs protect siRNA from RNase degradation while the complex is small enough to bypass the plant cell wall. Consequently, AuNCs enable gene knockdown with efficiencies of up 76.5 ± 5.9% and 76.1 ± 9.5% for GFP and ROQ1, respectively, with no observable toxicity. Our data suggest that AuNCs can deliver siRNA into intact plant cells for broad applications in plant biotechnology.

Additional Information

© 2021 American Chemical Society. Received 11 May 2021. Revised 17 June 2021. Published online 21 June 2021. Published in issue 14 July 2021. 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, an NSF CAREER award, the Dreyfus Foundation, the Chan-Zuckerberg foundation, and an FFAR New Innovator Award (to M.P.L.). Work at the Molecular Foundry was supported by the Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The authors acknowledge the support of the BASF-CARA program. We acknowledge support from the Keck Foundation (Grant 89208), and H.Z. acknowledges the support of the start-up fund from Jinan University (88016105). N.S.G. is supported by a Foundation for Food and Agriculture Research Fellowship. G.S.D. is supported by a Schlumberger Foundation Faculty for the Future Fellowship and the Resnick Sustainability Institute. The authors 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). Author Contributions: H.Z. and Y.C. contributed equally to this work. Yuhong C., H.Z., M.P.L., and P.Y. conceived the idea and designed the study. H.Z. and Yuhong C. performed the majority of the experiments and data analysis. H.Z., Yuhong C, S.C.-B., M.P.L., and P.Y. wrote or revised the manuscript. D.X. assisted in gold nanocluster synthesis. Yuan C. helped with plant seeding and maintenance. G.S.D and N.S.G. helped analyze the results. All authors edited the manuscript and approved the final version. The authors declare no competing financial interest.

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