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.
- Abbreviations
- ARF1
- AUXIN RESPONSE FACTOR1
- Cas9
- CRISPR-associated endonuclease 9
- CRISPR
- clustered regularly interspaced short palindromic repeats
- DSB
- double-strand break
- EF
- ELONGATION FACTOR1α
- HPT
- hygromycin phosphotransferase
- gRNA
- single guide RNA
- NAA
- 1-naphthalene acetic acid
- NHEJ
- non-homologous end joining
- NLS
- nuclear localization signal
- NOP1
- NOPPERABO1
- mALS
- mutated acetolactate synthase
- MMEJ
- microhomology-mediated end joining
- PAM
- protospacer adjacent motif
- PCR
- polymerase chain reaction
- RT-PCR
- reverse transcription polymerase chain reaction.