Viswan Anchu, Yamagishi Ayana, Hoshi Masamichi, Furuhata Yuichi, Kato Yoshio, Makimoto Natsumi, Takeshita Toshihiro, Kobayashi Takeshi, Iwata Futoshi, Kimura Mitsuhiro, Yoshizumi Takeshi, Nakamura Chikashi
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, Japan.
Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
Front Plant Sci. 2022 Jun 24;13:878059. doi: 10.3389/fpls.2022.878059. eCollection 2022.
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 leaves and (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 by quantifying the GUS (β-glucuronidase) expression that occurred by the recombination of the 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.
利用重组DNA对植物进行基因组编辑通常会导致外源DNA整合到宿主基因组中。为了防止产生不良的基因改变，人们期望将基因组编辑蛋白直接递送至植物组织中。然而，在目前大多数可用的方法中，基因组编辑蛋白的进入点无法控制，并且需要耗时的过程来筛选成功转移的样本。为了克服这些限制，我们考虑了一种基于新型微针阵列（MNA）的递送系统，其中针从基底表面水平排列，使其呈梳状结构。我们旨在将基因组编辑蛋白直接递送至叶组织的内层；栅栏组织、茎尖分生组织（SAM）的海绵组织和表皮下L2层，其中包括可分化为种系的细胞。宽度为2μm、长度为60μm的针阵列能够有效地插入拟南芥叶和大豆SAM中，且针不会弯曲或折断。该装置最初通过定量由位点重组产生的GUS（β-葡萄糖醛酸酶）表达，测试了将Cre重组酶递送至报告植物叶片中的情况。我们在每次插入时都观察到了GUS表达。此外，将靶向大豆SAM中PDS11/18基因的Cas9核糖核蛋白（RNP）直接递送，结果显示Cas9 RNP靶位点出现11bp的缺失。因此，该方法有效地将基因组编辑蛋白递送至植物组织中，并且对进入点进行了精确控制。
