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Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells

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A novel and robust lipofection-mediated transfection approach for the use of DNA-free Cas9/gRNA RNP for gene editing has demonstrated efficacy in plant cells.

Abstract

Precise genome editing has been revolutionized by CRISPR/Cas9 systems. DNA-based delivery of CRISPR/Cas9 is widely used in various plant species. However, protein-based delivery of the in vitro translated Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complex into plant cells is still in its infancy even though protein delivery has several advantages. These advantages include DNA-free delivery, gene-edited host plants that are not transgenic, ease of use, low cost, relative ease to be adapted to high-throughput systems, and low off-target cleavage rates. Here, we show a novel lipofection-mediated transfection approach for protein delivery of the preassembled Cas9/gRNA RNP into plant cells for genome editing. Two lipofection reagents, Lipofectamine 3000 and RNAiMAX, were adapted for successful delivery into plant cells of Cas9/gRNA RNP. A green fluorescent protein (GFP) reporter was fused in-frame with the C-terminus of the Cas9 protein and the fusion protein was successfully delivered into non-transgenic tobacco cv. ‘Bright Yellow-2’ (BY2) protoplasts. The optimal efficiencies for Lipofectamine 3000- and RNAiMAX-mediated protein delivery were 66% and 48%, respectively. Furthermore, we developed a biolistic method for protein delivery based on the known proteolistics technique. A transgenic tobacco BY2 line expressing an orange fluorescence protein reporter pporRFP was targeted for knockout. We found that the targeted mutagenesis frequency for our Lipofectamine 3000-mediated protein delivery was 6%. Our results showed that the newly developed lipofection-mediated transfection approach is robust for the use of the DNA-free Cas9/gRNA technology for genome editing in plant cells.

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Acknowledgements

The authors thank the Advance Research Projects Agency Energy (Award no. DE-AR0000331), Thermo Fisher. and the BioEnergy Science Center (BESC) for funding. The BESC was a U.S. Department of Energy (DOE) Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. We are also grateful for the funding from University of Tennessee and the Ivan Racheff endowment, and from North Carolina State University for the startup funds to the Liu laboratory. Research was enabled by Hatch grants. We thank Kacie Reynolds for her assistance with the miniprep work.

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Authors and Affiliations

  1. Department of Plant Sciences, University of Tennessee, Knoxville, TN, 37996, USA

    Wusheng Liu, Mary R. Rudis, Matthew H. Cheplick, Reginald J. Millwood, Christine A. Ondzighi-Assoume, Garrett A. Montgomery, Kellie P. Burris, Mitra Mazarei & Charles Neal Stewart Jr.

  2. Department of Horticultural Science, North Caroline State University, Raleigh, NC, 27607, USA

    Wusheng Liu

  3. Synthetic Biology Research and Development, Thermo Fisher Scientific, Carlsbad, CA, 92008, USA

    Jian-Ping Yang & Jonathan D. Chesnut

  4. Department of Agricultural and Environmental Sciences, Tennessee State University, Nashville, TN, 37209, USA

    Christine A. Ondzighi-Assoume

  5. Department of Food, Bioprocessing and Nutrition Sciences, North Caroline State University, Raleigh, NC, 27606, USA

    Kellie P. Burris

  6. Center for Agricultural Synthetic Biology, University of Tennessee Institute of Agriculture, Knoxville, TN, 37996, USA

    Charles Neal Stewart Jr.

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  1. Wusheng Liu
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  2. Mary R. Rudis
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Contributions

WL, JDC, JPY, and CNS conceived and designed the project. WL, MM, MHC, RJM, CAO, and GAM conducted the experiments, and collected and analyzed the data. KPB developed the transgenic BY2 cells. WL, MM, JDC, and CNS wrote the manuscript. All the authors read and approved the final manuscript.

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Correspondence to Wusheng Liu or Charles Neal Stewart Jr..

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Conflict of interest

The authors declare competing interests. The lipofection method is the subject of an invention disclosure and patent application at UTK. The University of Tennessee received funding from Thermo Fisher for the research and some of the authors are employees of the company.

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Communicated by Baochun Li.

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Liu, W., Rudis, M.R., Cheplick, M.H. et al. Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells. Plant Cell Rep 39, 245–257 (2020). https://doi.org/10.1007/s00299-019-02488-w

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