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直接将蛋白质递送至完整的拟南芥细胞中进行基因组编辑。

Direct protein delivery into intact Arabidopsis cells for genome engineering.

机构信息

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Higashi 1-1-1, Tsukuba, 305-8566, Japan.

Faculty of Agriculture, Takasaki University of Health and Welfare, 37-1 Nakaorui-machi, Takasaki-shi, Gunma, 370-0033, Japan.

出版信息

Sci Rep. 2024 Sep 29;14(1):22568. doi: 10.1038/s41598-024-72978-9.

DOI:10.1038/s41598-024-72978-9
PMID:39343787
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11439911/
Abstract

Intracellular delivery of biomolecules is a prerequisite for genetic techniques such as recombinant engineering and genome editing. Realizing the full potential of this technology requires the development of safe and effective methods for delivering protein tools into cells. In this study, we demonstrated the spontaneous internalization of exogenous proteins into intact cells and root tissue of whole plants of Arabidopsis thaliana. We termed this internalization phenomenon as protein Delivery Independent of Vehicles or Equipment (DIVE), which efficiently delivered genome engineering proteins including Cre recombinase and zinc-finger nucleases (ZFN) into plant cells. Using protein DIVE, we achieved less toxic protein delivery than electroporation with up to 94% efficiency in Arabidopsis cell culture and 19% genome modification in Arabidopsis plants that was maintained in regenerated tissue. This work illustrates the potential of protein DIVE for a wide range of applications, including genome engineering in plants.

摘要

生物分子的细胞内递送是重组工程和基因组编辑等基因技术的前提。为了充分发挥这项技术的潜力，需要开发安全有效的方法将蛋白工具递送到细胞内。在本研究中，我们证明了外源蛋白可自发地进入拟南芥完整细胞和根组织内。我们将这种内化现象称为蛋白非载体或设备依赖的内吞（protein Delivery Independent of Vehicles or Equipment，DIVE），它可以有效地将基因组工程蛋白（包括 Cre 重组酶和锌指核酸酶（ZFN））递送到植物细胞内。利用蛋白 DIVE，我们实现了比电穿孔更低毒性的蛋白递送，在拟南芥细胞培养中效率高达 94%，在再生组织中维持的拟南芥植物中的基因组修饰效率达到 19%。这项工作说明了蛋白 DIVE 在包括植物基因组工程在内的广泛应用中的潜力。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2d0c/11439911/2cc4a692f76b/41598_2024_72978_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2d0c/11439911/1766921f150c/41598_2024_72978_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2d0c/11439911/87b4b7173b77/41598_2024_72978_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2d0c/11439911/4d2b49594c2e/41598_2024_72978_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2d0c/11439911/5ddf7b686467/41598_2024_72978_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2d0c/11439911/c471fc3d5d37/41598_2024_72978_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2d0c/11439911/cc17057c0c58/41598_2024_72978_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2d0c/11439911/2cc4a692f76b/41598_2024_72978_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2d0c/11439911/1766921f150c/41598_2024_72978_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2d0c/11439911/87b4b7173b77/41598_2024_72978_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2d0c/11439911/4d2b49594c2e/41598_2024_72978_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2d0c/11439911/5ddf7b686467/41598_2024_72978_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2d0c/11439911/c471fc3d5d37/41598_2024_72978_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2d0c/11439911/cc17057c0c58/41598_2024_72978_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2d0c/11439911/2cc4a692f76b/41598_2024_72978_Fig7_HTML.jpg

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本文引用的文献

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A Method for Electroporation of Cre Recombinase Protein into Intact Cells.一种将Cre重组酶蛋白电穿孔导入完整细胞的方法。
Plants (Basel). 2023 Apr 12;12(8):1631. doi: 10.3390/plants12081631.
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Gene transfer to plants by electroporation: methods and applications.电穿孔法向植物转移基因：方法与应用。
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PLoS One. 2020 Jan 3;15(1):e0227477. doi: 10.1371/journal.pone.0227477. eCollection 2020.
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A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall.利用电穿孔法将 Cre 重组酶蛋白递送至具有完整细胞壁的培养拟南芥细胞中。
Sci Rep. 2019 Feb 15;9(1):2163. doi: 10.1038/s41598-018-38119-9.
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Genome Engineering and Agriculture: Opportunities and Challenges.基因组工程与农业：机遇与挑战
Prog Mol Biol Transl Sci. 2017;149:1-26. doi: 10.1016/bs.pmbts.2017.03.011. Epub 2017 May 3.
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Protein Delivery into Plant Cells: Toward Structural Biology.蛋白质导入植物细胞：迈向结构生物学
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