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

• Published in: PloS one Vol. 13; no. 10; p. e0205117
• Main Authors: Sugano, Shigeo S., Nishihama, Ryuichi, Shirakawa, Makoto, Takagi, Junpei, Matsuda, Yoriko, Ishida, Sakiko, Shimada, Tomoo, Hara-Nishimura, Ikuko, Osakabe, Keishi, Kohchi, Takayuki
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 31.10.2018; Public Library of Science (PLoS)
• Subjects: Biology and life sciences; Cell cycle; Combinatorial analysis; Complementation; CRISPR; Editing; Efficiency; Engineering and Technology; Feasibility studies; Flowers & plants; Functional analysis; Gene expression; Genetic analysis; Genetics; Genome editing; Genomes; Genomics; Kinases; Marchantia polymorpha; Mutation; Optimization; Proteins; Research and Analysis Methods; RNA polymerase; Sequences; Trends; Japan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0205117

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.