Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha

Shigeo S Sugano; Ryuichi Nishihama; Makoto Shirakawa; Junpei Takagi; Yoriko Matsuda; Sakiko Ishida; Tomoo Shimada; Ikuko Hara-Nishimura; Keishi Osakabe; Takayuki Kohchi

doi:10.1371/journal.pone.0205117

Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha

PLoS One. 2018 Oct 31;13(10):e0205117. doi: 10.1371/journal.pone.0205117. eCollection 2018.

Authors

Affiliations

¹ R-GIRO, Ritsumeikan University, Kusatsu, Shiga, Japan.
² JST, PRESTO, Kawaguchi, Saitama, Japan.
³ Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
⁴ Graduate School of Science, Kyoto University, Kyoto, Japan.
⁵ Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Tokushima, Japan.

Abstract

Marchantia polymorpha is one of the model species of basal land plants. Although CRISPR/Cas9-based genome editing has already been demonstrated for this plant, the efficiency was too low to apply to functional analysis. In this study, we show the establishment of CRISPR/Cas9 genome editing vectors with high efficiency for both construction and genome editing. Codon optimization of Cas9 to Arabidopsis achieved over 70% genome editing efficiency at two loci tested. Systematic assessment revealed that guide sequences of 17 nt or shorter dramatically decreased this efficiency. We also demonstrated that a combinatorial use of this system and a floxed complementation construct enabled conditional analysis of a nearly essential gene. This study reports that simple, rapid, and efficient genome editing is feasible with the series of developed vectors.

Publication types

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

MeSH terms

Arabidopsis / genetics
CRISPR-Cas Systems*
Gene Editing / methods*
Gene Knockout Techniques / methods
Genetic Vectors
Genome, Plant*
Immunoblotting
Marchantia / genetics*
Marchantia / metabolism
Mutation
Organisms, Genetically Modified
Phenotype

Grants and funding

This work was supported by the Grant-in-Aid for Specially Promoted Research to I.H-.N. [no. 22000014], by the Grant-in-Aid for Scientific Research on Innovative Area to T.K. [no. 25113009], by the Grant-in-Aid for Scientific Research (S) to T.K. [no. 17H07424], by the Grant-in-Aid for Scientific Research (C) to R.N. [no. 24570048], by the Grant-in-Aid for Young Scientists (B) to S.S.S. [no. 60726313], by the JST PRESTO Grant to S.S.S. [no. 11106], and by a donation from Otsuka Pharmaceuticals to Tokushima University. M.S. was supported by the Research Fellowship for Young Scientists [no. 24005453] and by the Postdoctoral Fellowships for Research Abroad from the Japan Society for the Promotion of Science (JSPS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.