Publications by authors named "Seiichi Toki"

The CRISPR-associated endonuclease Cas9 derived from prokaryotes is used as a genome editing, which targets specific genomic loci by single guide RNAs (sgRNAs). The eukaryotes, the target of genome editing, store their genome DNA in chromatin, in which the nucleosome is a basic unit. Despite previous structural analyses focusing on Cas9 cleaving free DNA, structural insights into Cas9 targeting of DNA within nucleosomes are limited, leading to uncertainties in understanding how Cas9 operates in the eukaryotic genome.

View Article and Find Full Text PDF

Base editors, CRISPR/Cas-based precise genome editing tools, enable base conversion at a target site without inducing DNA double-strand breaks. The genome editing targetable range is restricted by the requirement for protospacer adjacent motif (PAM) sequence. Cas9 derived from Streptococcus pyogenes (SpCas9)-most widely used for genome editing in many organisms-requires an NGG sequence adjacent to the target site as a PAM.

View Article and Find Full Text PDF

Gene targeting (GT) is a precise genome editing tool to achieve desired modification of a target gene, e.g., introduction of point mutations, knock-in of a reporter gene, or swapping of a functional domain, through homologous recombination.

View Article and Find Full Text PDF

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR-Cas) is an adaptive prokaryote immune system against foreign DNA/RNA that is now applied widely to genome editing. A miniature Cas, CRISPR-Cas12f, is one-half to one-third the size of the CRISPR-Cas9 that is commonly used in genome editing experiments in many organisms, including higher plants. The compactness of CRISPR-Cas12f is expected to be advantageous in terms of vector construction and transformation frequency.

View Article and Find Full Text PDF
Article Synopsis
  • Virus vectors like potato virus X (PVX) can deliver genome-editing tools such as SpCas9 into plant cells, but the large size of SpCas9 limits its effectiveness in systemic leaves.
  • Smaller Cas variants like AsCas12f have been engineered to improve genome-editing activity, and the modified AsCas12f demonstrated enhanced editing frequencies in rice.
  • The study found that a PVX vector with AsCas12f allowed efficient genome editing not only in inoculated leaves but also in systemic leaves, achieving over 60% regeneration of genome-edited shoots without foreign DNA.
View Article and Find Full Text PDF

Conventional plant gene editing requires laborious tissue-culture-mediated transformation, which restricts the range of applicable plant species. In this study, we developed a heritable and tissue-culture-free gene editing method in Nicotiana benthamiana using tobacco ringspot virus (TRSV) as a vector for in planta delivery of Cas9 and single-guide RNA (sgRNA) to shoot apical meristems. Agrobacterium-mediated inoculation of the TRSV vector induced systemic and heritable gene editing in Nicotiana benthamiana PHYTOENE DESATURASE.

View Article and Find Full Text PDF

Transposons are mobile genetic elements that can move to a different position within a genome or between genomes. They have long been used as a tool for genetic engineering, including transgenesis, insertional mutagenesis, and marker excision, in a variety of organisms. The transposon derived from the cabbage looper moth is one of the most promising transposon tools ever identified because has the advantage that it can transpose without leaving a footprint at the excised site.

View Article and Find Full Text PDF
Article Synopsis
  • The text discusses the challenges of modifying traits in a popular ornamental plant due to its complex genome structure and self-incompatibility.
  • Genome editing technology is identified as a promising method for modifying these traits, but it must ensure trait stability for commercial use.
  • The study explores the use of a transposon system to remove genome editing tools after successful trait modification, demonstrating this with a visible luminescence marker.
View Article and Find Full Text PDF

Sugi (Cryptomeria japonica D. Don) is an economically important coniferous tree in Japan. However, abundant sugi pollen grains are dispersed and transported by the wind each spring and cause a severe pollen allergy syndrome (Japanese cedar pollinosis).

View Article and Find Full Text PDF

Transposition of transposable elements affect expression levels, splicing and epigenetic status, and function of genes located in, or near, the inserted/excised locus. For example, in grape, presence of the Gret1 retrotransposon in the promoter region of the VvMYBA1a allele at the VvMYBA1 locus suppress the expression of the VvMYBA1 transcription factor gene for the anthocyanin biosynthesis and this transposon insertion is responsible for the green berry skin color of Vitis labrascana, 'Shine Muscat', a major grape cultivar in Japan. To prove that transposons in grape genome can be removed by genome editing, we focused on Gret1 in the VvMYBA1a allele as a target of CRISPR/Cas9 mediated transposon removal.

View Article and Find Full Text PDF

Cas9 derived from (SpCas9) is used widely in genome editing using the CRISPR-Cas system due to its high activity, but is a relatively large molecule (1,368 amino acid (a.a.) residues).

View Article and Find Full Text PDF

Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT.

View Article and Find Full Text PDF
Article Synopsis
  • Magnesium homeostasis is vital for biological processes, but the mechanisms regulating Mg levels in rice (Oryza sativa) are not well understood.
  • Through a forward genetic approach, researchers identified a rice mutant called LMGC1 that has reduced Mg levels in leaves and increased levels in roots, indicating problems with Mg uptake and transport.
  • The causal gene for the low Mg phenotype, identified as OsRZF1, is a nuclear protein that likely plays a key role in maintaining Mg homeostasis in rice.
View Article and Find Full Text PDF

Land plants are constantly exposed to environmental stresses and have developed complicated defense systems, including DNA damage response (DDR) and DNA repair systems, to protect plant cells. In Arabidopsis (Arabidopsis thaliana), the transcription factor SUPPRESSOR OF GAMMA RESPONSE1 (SOG1) plays a key role in DDR. Here, we focus on DDR in rice (Oryza sativa)-thought to be a simpler system compared with Arabidopsis due to lack of induction of the endocycle even under DNA damage stress.

View Article and Find Full Text PDF

Genome editing technology can be used for gene engineering in many organisms. A target metabolite can be fortified by the knockout and modification of target genes encoding enzymes involved in catabolic and biosynthesis pathways, respectively, via genome editing technology. Genome editing is also applied to genes encoding proteins other than enzymes, such as chaperones and transporters.

View Article and Find Full Text PDF

Flowering is an important biological process through which plants determine the timing of reproduction. In rice, florigen mRNA is induced more strongly when the day length is shorter than the critical day length through recognition of 30-min differences in the photoperiod. , which encodes a CCT-domain protein unique to monocots, has been identified as a key floral repressor in rice, and , a rice ortholog of the floral activator , is another key floral regulator gene.

View Article and Find Full Text PDF

The CRISPR/Cas12a (Cpf1) system utilizes a thymidine-rich protospacer adjacent motif (PAM) and generates DNA ends with a 5' overhang. These properties differ from those of CRISPR/Cas9, making Cas12a an attractive alternative in the CRISPR toolbox. However, genome editing efficiencies of Cas12a orthologs are generally lower than those of SpCas9 and depend on their target sequences.

View Article and Find Full Text PDF

Homologous recombination-mediated genome editing, also called gene targeting (GT), is an essential technique that allows precise modification of a target sequence, including introduction of point mutations, knock-in of a reporter gene, and/or swapping of a functional domain. However, due to its low frequency, it has been difficult to establish GT approaches that can be applied widely to a large number of plant species. We have developed a simple and universal clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated DNA double-strand break (DSB)-induced GT system using an all-in-one vector comprising a CRISPR/Cas9 expression construct, selectable marker, and GT donor template.

View Article and Find Full Text PDF

Facing the challenges of the world's food sources posed by a growing global population and a warming climate will require improvements in plant breeding and technology. Enhancing crop resiliency and yield via genome engineering will undoubtedly be a key part of the solution. The advent of new tools, such as CRIPSR/Cas, has ushered in significant advances in plant genome engineering.

View Article and Find Full Text PDF

Limitations for the application of genome editing technologies on elite wheat ( L.) varieties are mainly due to the dependency on culture and regeneration capabilities. Recently, we developed an particle bombardment (iPB) method which has increased process efficiency since no culture steps are required to create stably genome-edited wheat plants.

View Article and Find Full Text PDF

Genome-editing technologies consisting of targeted mutagenesis and gene targeting enable us to modify genes of interest rapidly and precisely. The discovery in 2012 of CRISPR/Cas9 systems and their development as sequence-specific nucleases has brought about a paradigm shift in biology. Initially, CRISPR/Cas9 was applied in targeted mutagenesis to knock out a target gene.

View Article and Find Full Text PDF

Targeted mutagenesis via CRISPR/Cas9 is now widely used, not only in model plants but also in agriculturally important crops. However, in vegetative crop propagation, CRISPR/Cas9 expression cassettes cannot be segregated out in the resulting progenies, but must nevertheless be eliminated without leaving unnecessary sequences in the genome. To this end, we designed a piggyBac-mediated transgenesis system for the temporary expression of CRISPR/Cas9 in plants.

View Article and Find Full Text PDF

Here, we describe a protocol for producing multiple recessive mutants via genome editing in hexaploid wheat () cv. Fielder. Using -delivered CRISPR/Cas9 and three sub-genome-specific primer sets, all possible combinations of single, double, and triple transgene-free mutants can be generated.

View Article and Find Full Text PDF

A PHP Error was encountered

Severity: Warning

Message: fopen(/var/lib/php/sessions/ci_session72okg7582lan0kd6q6gfktgiv9c42m8j): Failed to open stream: No space left on device

Filename: drivers/Session_files_driver.php

Line Number: 177

Backtrace:

File: /var/www/html/index.php
Line: 316
Function: require_once

A PHP Error was encountered

Severity: Warning

Message: session_start(): Failed to read session data: user (path: /var/lib/php/sessions)

Filename: Session/Session.php

Line Number: 137

Backtrace:

File: /var/www/html/index.php
Line: 316
Function: require_once