Date Published: October 31, 2018
Publisher: Public Library of Science
Author(s): Shigeo S. Sugano, Ryuichi Nishihama, Makoto Shirakawa, Junpei Takagi, Yoriko Matsuda, Sakiko Ishida, Tomoo Shimada, Ikuko Hara-Nishimura, Keishi Osakabe, Takayuki Kohchi, Hiroshi Ezura.
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.
The clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated endonuclease 9 (Cas9)-based genome editing system is a groundbreaking technology in molecular genetics, which enables alterations of target sequences in the genome [1, 2]. The system consists of two components: a Cas9 protein, which has an RNA-guided endonuclease activity, and a single guide RNA (gRNA), which specifies a target sequence within the genome. The Cas9 protein from Streptococcus pyogenes binds to the DNA sequence “NGG,” which is known as the protospacer adjacent motif (PAM) sequence. The interaction between a Cas9 protein and a PAM sequence induces the interaction between a gRNA and its target DNA sequence. If a sufficient length of the gRNA matches with the target sequence, the nuclease domains of Cas9 become capable of cutting the phosphodiester bonds on both sides of the strands, which are located 3 bp upstream of the PAM sequence . Once a double-strand break (DSB) occurs, the error-prone non-homologous end joining (NHEJ) repair pathway is activated and sometimes introduces indels or base substitutions randomly at the target site, which could result in disruption of the target locus with various alleles.
Using the Atco-Cas9 expression cassette, we successfully optimized the CRISPR/Cas9-based genome editing system for M. polymorpha and improved the efficiency to a degree that does not require target gene-based phenotypic selection. This is consistent with a previous report that codon optimization of Cas9 lead to significant improvement in efficiency . In fact, a higher accumulation level of Cas9 protein was observed with the Atco-Cas9 expression cassette, compared with the hCas9 one (S8 Fig). In the pMpGE010/011 system, over 70% of transformants underwent targeted mutagenesis, shown by the two gRNAs ARF1_1 and NOP1_1 (Figs 1 and 2). These results indicate that the pMpGE010/011 vectors are highly reliable for obtaining genome-edited lines in M. polymorpha. Since the GC content of the M. polymorpha genome is 49.8% , Cas9 target sites with NGG PAM sequences can be found at high probability, and the selection of gRNA target sites with small numbers of off-target sites is possible. Taken together, we conclude that the pMpGE010/pMpGE011 system is feasible to use for functional genetics in M. polymorpha with likely avoidance of off-target effects.