Microneedle Array-Assisted, Direct Delivery of Genome-Editing Proteins Into Plant Tissue

Anchu Viswan; Ayana Yamagishi; Masamichi Hoshi; Yuichi Furuhata; Yoshio Kato; Natsumi Makimoto; Toshihiro Takeshita; Takeshi Kobayashi; Futoshi Iwata; Mitsuhiro Kimura; Takeshi Yoshizumi; Chikashi Nakamura

doi:10.3389/fpls.2022.878059

Microneedle Array-Assisted, Direct Delivery of Genome-Editing Proteins Into Plant Tissue

Front Plant Sci. 2022 Jun 24:13:878059. doi: 10.3389/fpls.2022.878059. eCollection 2022.

Authors

Anchu Viswan^{1

2}, Ayana Yamagishi^{1

2}, Masamichi Hoshi^{1

2}, Yuichi Furuhata³, Yoshio Kato³, Natsumi Makimoto⁴, Toshihiro Takeshita⁴, Takeshi Kobayashi⁴, Futoshi Iwata⁵, Mitsuhiro Kimura⁶, Takeshi Yoshizumi⁶, Chikashi Nakamura^{1

2}

Affiliations

¹ Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, Japan.
² Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
³ Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
⁴ Sensing System Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
⁵ Graduate School of Medical Photonics, Shizuoka University, Hamamatsu, Japan.
⁶ Faculty of Agriculture, Takasaki University of Health and Welfare, Takasaki, Japan.

Abstract

Genome editing in plants employing recombinant DNA often results in the incorporation of foreign DNA into the host genome. The direct delivery of genome-editing proteins into plant tissues is desired to prevent undesirable genetic alterations. However, in most currently available methods, the point of entry of the genome-editing proteins cannot be controlled and time-consuming processes are required to select the successfully transferred samples. To overcome these limitations, we considered a novel microneedle array (MNA)-based delivery system, in which the needles are horizontally aligned from the substrate surface, giving it a comb-like configuration. We aimed to deliver genome-editing proteins directly into the inner layers of leaf tissues; palisade, the spongy and subepidermal L2 layers of the shoot apical meristem (SAM) which include cells that can differentiate into germlines. The array with needles 2 μm wide and 60 μm long was effective in inserting into Arabidopsis thaliana leaves and Glycine max (L.) Merr. (soybeans) SAM without the needles buckling or breaking. The setup was initially tested for the delivery of Cre recombinase into the leaves of the reporter plant A. thaliana by quantifying the GUS (β-glucuronidase) expression that occurred by the recombination of the loxP sites. We observed GUS expression at every insertion. Additionally, direct delivery of Cas9 ribonucleoprotein (RNP) targeting the PDS11/18 gene in soybean SAM showed an 11 bp deletion in the Cas9 RNP target site. Therefore, this method effectively delivered genome-editing proteins into plant tissues with precise control over the point of entry.

Keywords: Arabidopsis leaf; Cas9; Cre recombinase; direct delivery; genome editing; microneedle array (MNA); soybean SAM.