Efficient genome editing by controlled release of Cas9 ribonucleoprotein in plant cytosol using polymer-modified microneedle array

Anchu Viswan; Chiaki Yoshikawa; Ayana Yamagishi; Yuichi Furuhata; Yoshio Kato; Tomohiko Yamazaki; Chikashi Nakamura

doi:10.1016/j.bbrc.2023.149179

Efficient genome editing by controlled release of Cas9 ribonucleoprotein in plant cytosol using polymer-modified microneedle array

Biochem Biophys Res Commun. 2023 Dec 17:686:149179. doi: 10.1016/j.bbrc.2023.149179. Epub 2023 Oct 29.

Authors

Anchu Viswan¹, Chiaki Yoshikawa², Ayana Yamagishi¹, Yuichi Furuhata³, Yoshio Kato³, Tomohiko Yamazaki², Chikashi Nakamura⁴

Affiliations

¹ Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan; Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
² Research Center of Functional Materials, National Institute for Materials Science, Tsukuba, Ibaraki, Japan.
³ Research Center for Macromolecules & Biomaterials, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
⁴ Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan; Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan. Electronic address: chikashi-nakamura@aist.go.jp.

PMID: 37922572
DOI: 10.1016/j.bbrc.2023.149179

Abstract

Direct delivery of genome-editing proteins into plant tissues could be useful in obtaining DNA-free genome-edited crops obviating the need for backcrossing to remove vector-derived DNA from the host genome as in the case of genetically modified organisms generated using DNA vector. Previously, we successfully delivered Cas9 ribonucleoprotein (RNP) into plant tissue by inserting microneedle array (MNA) physisorbed with Cas9 RNPs. Here, to enhance protein delivery and improve genome-editing efficiency, we introduced a bioactive polymer DMA/HPA/NHS modification to the MNA, which allowed strong bonding between the proteins and MNA. Compared with other modifying agents, this MNA modification resulted in better release of immobilized protein in a plant cytosol-mimicking environment. The delivery of Cas9 RNPs in Arabidopsis thaliana reporter plants was improved from 4 out of 17 leaf tissues when using unmodified MNAs to 9 out of 17 when using the polymer-modified MNAs. Further improvements in delivery efficiency can be envisaged by optimizing the polymer modification conditions, which could have significant implications for the development of more effective plant genome editing techniques.

Keywords: Arabidopsis leaf; Bioactive polymer; Cas9; Genome editing; Microneedle array; Silicon.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems*
Cytosol / metabolism
DNA
Delayed-Action Preparations
Gene Editing* / methods
Genome, Plant / genetics
Ribonucleoproteins / genetics
Ribonucleoproteins / metabolism

Substances

Ribonucleoproteins
Delayed-Action Preparations
DNA