TY - JOUR T1 - Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in <em>Marchantia polymorpha</em> JF - bioRxiv DO - 10.1101/277350 SP - 277350 AU - Shigeo S. Sugano AU - Ryuichi Nishihama AU - Makoto Shirakawa AU - Junpei Takagi AU - Yoriko Matsuda AU - Sakiko Ishida AU - Tomoo Shimada AU - Ikuko Hara-Nishimura AU - Keishi Osakabe AU - Takayuki Kohchi Y1 - 2018/01/01 UR - http://biorxiv.org/content/early/2018/03/14/277350.abstract N2 - 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.AbbreviationsARF1AUXIN RESPONSE FACTOR1Cas9CRISPR-associated endonuclease 9CRISPRclustered regularly interspaced short palindromic repeatsDSBdouble-strand breakEFELONGATION FACTOR1αHPThygromycin phosphotransferasegRNAsingle guide RNANAA1-naphthalene acetic acidNHEJnon-homologous end joiningNLSnuclear localization signalNOP1NOPPERABO1mALSmutated acetolactate synthaseMMEJmicrohomology-mediated end joiningPAMprotospacer adjacent motifPCRpolymerase chain reactionRT-PCRreverse transcription polymerase chain reaction. ER -