title: pKAMA-ITACHI Vectors for Highly Efficient CRISPR/Cas9-Mediated Gene Knockout in Arabidopsis thaliana.
abstract: The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is widely used as a tool for genome engineering in various organisms. A complex consisting of Cas9 and single guide RNA (sgRNA) induces a DNA double-strand break in a sequence-specific manner, resulting in knockout. Some binary vectors for CRISPR/Cas9 in plants have been reported, but there is a problem with low efficiency. Here, we present a newly developed, highly efficient CRISPR/Cas9 vector for Arabidopsis thaliana, pKAMA-ITACHI Red (pKIR), harboring the RIBOSOMAL PROTEIN S5 A (RPS5A) promoter to drive Cas9. The RPS5A promoter maintains high constitutive expression at all developmental stages starting from the egg cell and including meristematic cells. Even in the T1 generation, pKIR induced null phenotypes in some genes: PHYTOENE DESATURASE 3 (PDS3), AGAMOUS (AG) and DUO POLLEN 1 (DUO1). Mutations induced by pKIR were carried in the germ cell line of the T1 generation. Surprisingly, in some lines, 100% of the T2 plants had the adh1 (ALCOHOL DEHYDROGENASE 1) null phenotype, indicating that pKIR strongly induced heritable mutations. Cas9-free T2 mutant plants were obtained by removing T2 seeds expressing a fluorescent marker in pKIR. Our results suggest that the pKIR system is a powerful molecular tool for genome engineering in Arabidopsis.
authors.affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, Japan.|JST ERATO Higashiyama Live-Holonics Project, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Tsutsui
authors.affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, Japan.|JST ERATO Higashiyama Live-Holonics Project, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, Japan.|Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Higashiyama
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D000072669:Gene Editing / Q000379:methods*; D055786:Gene Knockout Techniques / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D009154:Mutation; D030821:Plants, Genetically Modified; D015203:Reproducibility of Results; D012689:Sequence Homology, Nucleic Acid
keywords: Arabidopsis thaliana; CRISPR/Cas9; Cas9-free isolation; Genome engineering; Knockout