Precise Base Substitution Using CRISPR/Cas-Mediated Base Editor in Rice

Masaki Endo; Katsuya Negishi; Seiichi Toki

doi:10.1007/978-1-0716-4204-7_11

Precise Base Substitution Using CRISPR/Cas-Mediated Base Editor in Rice

Methods Mol Biol. 2025:2869:101-111. doi: 10.1007/978-1-0716-4204-7_11.

Authors

Masaki Endo¹, Katsuya Negishi², Seiichi Toki^{3

4

5

6}

Affiliations

¹ Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan. mendo@affrc.go.jp.
² Institute of Fruit Tree and Tea Science, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
³ Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
⁴ Graduate School of Nanobioscience, Yokohama City University, Yokohama, Japan.
⁵ Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan.
⁶ Faculty of Agriculture, Department of Life Science, Ryukoku University, Kyoto, Japan.

PMID: 39499471
DOI: 10.1007/978-1-0716-4204-7_11

Abstract

Base editors, CRISPR/Cas-based precise genome editing tools, enable base conversion at a target site without inducing DNA double-strand breaks. The genome editing targetable range is restricted by the requirement for protospacer adjacent motif (PAM) sequence. Cas9 derived from Streptococcus pyogenes (SpCas9)-most widely used for genome editing in many organisms-requires an NGG sequence adjacent to the target site as a PAM. Then, engineered and natural Cas variants with altered PAM recognition are used for base editor to expand the flexibility of base substitution position. In this chapter, we describe a protocol for base editing based on SpCas9-NG, which is a rationally engineered SpCas9 variant that can recognize relaxed NG PAMs.

Keywords: Adenine base editors; Agrobacterium-mediated transformation; Base substitution; CRISPR/Cas; Cytosine base editors.

MeSH terms

CRISPR-Associated Protein 9 / genetics
CRISPR-Associated Protein 9 / metabolism
CRISPR-Cas Systems*
Gene Editing* / methods
Genome, Plant
Oryza* / genetics
Plants, Genetically Modified / genetics
RNA, Guide, CRISPR-Cas Systems / genetics
Streptococcus pyogenes / genetics

Substances

CRISPR-Associated Protein 9
RNA, Guide, CRISPR-Cas Systems