Targeted genome modification technologies are key tools for functional genomics. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 system (CRISPR/Cas9) is an emerging technology for targeted genome modification. The CRISPR/Cas9 system consists of a short guide RNA (gRNA), which specifies the target genome sequence, and the Cas9 protein, which has endonuclease activity. The CRISPR/Cas9 system has been applied to model animals and flowering plants, including rice, sorghum, wheat, tobacco and Arabidopsis. Here, we report the application of CRISPR/Cas9 to targeted mutagenesis in the liverwort Marchantia polymorpha L., which has emerged as a model species for studying land plant evolution. The U6 promoter of M. polymorpha was identified and cloned to express the gRNA. The target sequence of the gRNA was designed to disrupt the gene encoding auxin response factor 1 (ARF1) in M. polymorpha. Using Agrobacterium-mediated transformation, we isolated stable mutants in the gametophyte generation of M. polymorpha. CRISPR/Cas9-based site-directed mutagenesis in vivo was achieved using either the Cauliflower mosaic virus 35S or M. polymorpha EF1α promoter to express Cas9. Isolated mutant individuals showing an auxin-resistant phenotype were not chimeric. Moreover, stable mutants were produced by asexual reproduction of T1 plants. Multiple arf1 alleles were easily established using CRIPSR/Cas9-based targeted mutagenesis. Our results provide a rapid and simple approach for molecular genetics in M. polymorpha, and raise the possibility that CRISPR/Cas9 may be applied to a wide variety of plant species.

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 cases of 29 patients with cervical myelopathy, who had been treated by anterior spine fusion, were reviewed. The relationship between pre- and postoperative magnetic resonance (MR) images was investigated with special reference to increased signal intensity in the spinal cord on the T2-weighted images and the relevance of this finding to clinical conditions. Preoperatively, there were areas of increased signal intensity in 12 patients whereas there were no areas of increased signal intensity in the other 17. The lesions were not clearly demonstrated on T1-weighted images. The pre- and postoperative clinical condition of the patients whose preoperative MR images showed areas of increased signal intensity in the spinal cord on T2-weighted images was worse than that of the patients who did not have areas of increased signal intensity. Of the 12 patients with regions of increased signal intensity preoperatively, five showed decreased signal intensity postoperatively compared to the preoperative levels and seven had no change. The postoperative recovery of the five patients who showed decreased signal intensity postoperatively was better than that of the seven patients who exhibited no change. The areas of increased MR signal in the spinal cord might be due to edema, cord gliosis, demyelination, or microcavities.

19Marchantia polymorpha is one of the model species of basal land plants. Although 20 CRISPR/Cas9-based genome editing has already been demonstrated for this plant, the 21 efficiency was too low to apply to functional analysis. In this study, we show the 22 establishment of CRISPR/Cas9 genome editing vectors with high efficiency for both 23 construction and genome editing. Codon optimization of Cas9 to Arabidopsis 24 achieved over 70% genome editing efficiency at two loci tested. Systematic 25 assessment revealed that guide sequences of 17 nt or shorter dramatically decreased 26 this efficiency. We also demonstrated that a combinatorial use of this system and a 27 floxed complementation construct enabled conditional analysis of a nearly essential 28gene. This study reports that simple, rapid, and efficient genome editing is feasible 29 with the series of developed vectors. 30 31 32 Abbreviations: ARF1, AUXIN RESPONSE FACTOR1; Cas9, CRISPR-associated endonuclease 9; 33 CRISPR, clustered regularly interspaced short palindromic repeats; DSB, double-strand break; EF, 34 ELONGATION FACTOR1α; HPT, hygromycin phosphotransferase; gRNA, single guide RNA; 35 NAA, 1-naphthalene acetic acid; NHEJ, non-homologous end joining; NLS, nuclear localization 36 signal; NOP1, NOPPERABO1; mALS, mutated acetolactate synthase; MMEJ, microhomology-37 mediated end joining; PAM, protospacer adjacent motif; PCR, polymerase chain reaction; RT-38 PCR, reverse transcription polymerase chain reaction.39 40 such haploid generation-dominant plant species are free from the transheterozygosity 66 issues associated with diploidy or polyploidy [12][13][14], allowing isolation of pure 67 mutant lines for analysis with relative ease. In the meanwhile, regardless of the ploidy, 68 but especially for haploid species, genome editing techniques cannot be simply 69 applied to essential genes as this leads to lethality; conditional approaches are 70 required. 71The liverwort Marchantia polymorpha is an emerging model species of land 72 plants for studying plant evolution and gene function [15]. M. polymorpha has good 73 features for the application of reverse genetics. Most vascular plants and mosses are 74 known to have experienced two or more whole genome duplication events, which 75 makes it difficult to analyze gene functions due to the presence of paralogous genes. 76 Sequencing of the M. polymorpha genome revealed no sign of a whole genome 77 duplication and accordingly there is low genetic redundancy in most regulatory genes, 78 such as transcription factors and signaling components [16]. In addition, non-chimeric 79 individuals can be easily obtained and propagated via gemmae that are derived from 80 single cells by asexual reproduction in M. polymorpha [17], which accelerates 81 transgenic experiments [18]. A variety of tools for molecular genetic experiments 82 have been developed for M. polymorpha [18], such as high-efficiency transformation 83 methods [19-21], a homologous recombination-mediated gene targeting method [22], 84 a systematic set of ...

Light regulates various aspects of development throughout the life cycle of sessile land plants. Photoreceptors, such as the red (R) and far-red (FR) light receptors phytochromes, play pivotal roles in modulating developmental programs. Reflecting high developmental plasticity, plants can regenerate tissues, organs, and whole bodies from varieties of cells. Among land plants, bryophytes exhibit extraordinary competency of regeneration under hormone-free conditions. As an environmental factor, light plays critical roles in regeneration of bryophytes. However, how light regulates regeneration remains unknown. Here we show that using the liverwort Marchantia polymorpha, which contains a single phytochrome gene, the phytochrome regulates re-entry into the cell cycle and cell shape in newly regenerating tissues. Our morphological and cytological observations revealed that S-phase entry of G1-arrested epidermal cells around the midrib on the ventral surface of thallus explants was greatly retarded in the dark or under phytochrome-inactive R/FR cycle irradiation conditions, where, nevertheless, small, laterally narrow regenerants were eventually formed. Thus, consistent with earlier descriptions published over a century ago, light is not essential for, but exerts profound effects on regeneration in M. polymorpha. Ventral cells in regenerants grown under R/FR cycle conditions were longer and narrower than those under R cycle. Expression of a constitutively active mutant of M. polymorpha phytochrome allowed regeneration of well grown, widely expanded thalli even in the dark when sugar was supplied, further demonstrating that the phytochrome signal promotes cell proliferation, which is rate-limited by sucrose availability. Similar effects of R and FR irradiation on cell division and elongation were observed in sporelings as well. Thus, besides activation of photosynthesis, major roles of R in regeneration of M. polymorpha are to facilitate proliferation of rounder cells through the phytochrome by mechanisms that are likely to operate in the sporeling.