Structural insights into how Cas9 targets nucleosomes.

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.

Precise Base Substitution Using CRISPR/Cas-Mediated Base Editor in Rice.

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.

CRISPR/Cas9-mediated disruption of CjACOS5 confers no-pollen formation on sugi trees (Cryptomeria japonica D. Don).

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).

Targeted deletion of grape retrotransposon associated with fruit skin color via CRISPR/Cas9 in Vitis labrascana 'Shine Muscat'.

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.

A MAPKKK gene from rice, RBG1res, confers resistance to Burkholderia glumae through negative regulation of ABA.

Burkholderia glumae causes bacterial seedling rot (BSR) of rice and is a threat to a consistent food supply. When previously screening for resistance against B. glumae in the resistant cultivar Nona Bokra (NB) versus the susceptible cultivar Koshihikari (KO), we detected a gene, Resistance to Burkholderia glumae 1 (RBG1), at a quantitative trait locus (QTL).

Genome editing by introduction of Cas9/sgRNA into plant cells using temperature-controlled atmospheric pressure plasma.

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.

Current status and perspectives for endoscopic diagnosis of superficial nonampullary duodenal epithelial tumors.

In recent years, there have been significant advances in the endoscopic resection (ER) procedures of superficial nonampullary duodenal epithelial tumors (SNADETs). A preoperative endoscopic diagnosis is thus deemed necessary in determining the indication for subsequent ER. For the histologic and endoscopic diagnosis of SNADETs, understanding the mucin phenotype is inevitable.

Oxicam-type nonsteroidal anti-inflammatory drugs enhance -mediated transient transformation in plants.

-mediated transformation is a key innovation for plant breeding, and routinely used in basic researches and applied biology. However, the transformation efficiency is often the limiting factor of this technique. In this study, we discovered that oxicam-type nonsteroidal anti-inflammatory drugs, including tenoxicam (TNX), increase the efficiency of -mediated transient transformation.

Enhanced FnCas12a-Mediated Targeted Mutagenesis Using crRNA With Altered Target Length in Rice.

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.

The mucin phenotype does not affect the endoscopic resection outcome of non-ampullary duodenal epithelial tumors.

Some studies have reported an association between clinicopathological features and mucin phenotypes of non-ampullary duodenal epithelial tumors (NADETs). However, the association between clinical outcomes of endoscopic resection (ER) and mucin phenotypes has not been elucidated. The aim of this retrospective study was to analyze clinical outcomes of ER of NADETs with reference to mucin phenotypes.

Regulation of germination by targeted mutagenesis of grain dormancy genes in barley.

High humidity during harvest season often causes pre-harvest sprouting in barley (Hordeum vulgare). Prolonged grain dormancy prevents pre-harvest sprouting; however, extended dormancy can interfere with malt production and uniform germination upon sowing. In this study, we used Cas9-induced targeted mutagenesis to create single and double mutants in QTL FOR SEED DORMANCY 1 (Qsd1) and Qsd2 in the same genetic background.

Prognostic nutritional index is an independent prognostic factor for older patients aged ≥ 85 years treated by gastric endoscopic submucosal dissection.

Background: Clinical outcomes and prognostic factors for survival after endoscopic submucosal dissection (ESD) in older patients aged ≥ 85 years with early gastric cancer (EGC) are not well defined. The aim of this study was to investigate the clinical outcomes and prognostic factors for survival after ESD in older patients aged ≥ 85 years with EGC.

Methods: Clinical outcomes of 70 patients aged ≥ 85 years with EGC treated with ESD were evaluated retrospectively.

Resectability of underwater endoscopic mucosal resection for duodenal tumor: A single-center, retrospective pilot study.

Background And Aim: Underwater endoscopic mucosal resection (U-EMR) has been attracting much attention as treatment for patients with nonampullary duodenal epithelial tumors (NADETs). We aim to compare treatment outcomes, including submucosal resectability, between patients undergoing U-EMR and conventional endoscopic mucosal resection (C-EMR) for NADET.

Methods: We conducted a retrospective review of 38 patients with NADET treated by U-EMR or C-EMR.

Genome Editing in Commercial Wheat Varieties.

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.

Mutation of the imprinted gene OsEMF2a induces autonomous endosperm development and delayed cellularization in rice.

In angiosperms, endosperm development comprises a series of developmental transitions controlled by genetic and epigenetic mechanisms that are initiated after double fertilization. Polycomb repressive complex 2 (PRC2) is a key component of these mechanisms that mediate histone H3 lysine 27 trimethylation (H3K27me3); the action of PRC2 is well described in Arabidopsis thaliana but remains uncertain in cereals. In this study, we demonstrate that mutation of the rice (Oryza sativa) gene EMBRYONIC FLOWER2a (OsEMF2a), encoding a zinc-finger containing component of PRC2, causes an autonomous endosperm phenotype involving proliferation of the central cell nuclei with separate cytoplasmic domains, even in the absence of fertilization.

Investigation of clomazone-tolerance mechanism in a long-grain cultivar of rice.

Background: Clomazone is a potent herbicide for controlling weeds that have evolved resistance to other herbicides due to its unique mode of action. Clomazone is used in rice cultivation, but is limited to long-grain cultivars because other cultivars are highly sensitive to it. In this study, we investigated the mechanism of clomazone tolerance in a long-grain cultivar.

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T generation.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system.

Protective effect of proton pump inhibitors and potassium competitive acid blockers against post-gastric endoscopic submucosal dissection bleeding: a single-center, propensity score-matched analysis.

Objectives: Both potassium-competitive acid blockers (P-CABs) and proton pump inhibitors (PPIs) are known to be protective against bleeding after gastric endoscopic dissection (ESD) for early gastric cancers. The aim was to compare the effect of PPI and P-CAB treatment against bleeding after gastric ESD.

Materials And Methods: This was a single-center, retrospective analysis.

Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis.

Background: Soybean (Glycine max) is a major protein crop, because soybean protein has an amino acid score comparable to that of beef and egg white. However, many allergens have been identified among soybean proteins. A decrease in allergenic protein levels would be useful for expanding the market for soybean proteins and processed foods.

Protocol for Genome Editing to Produce Multiple Mutants in Wheat.

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.

Immunohistochemical Analysis of Mismatch Repair Gene Proteins in Early Gastric Cancer Based on Microsatellite Status.

Background: Microsatellite instability (MSI) is a major pathway involved in gastric carcinogenesis and is observed in 10-20% of early gastric cancers (EGCs). Early detection of EGCs with an MSI-high phenotype would be useful for elucidating the mechanisms of gastric carcinogenesis and improving outcomes in patients with GC.

Objective: We explored the usefulness of immunohistochemical expression of mismatch repair (MMR) proteins, including MLH1, PMS2, MSH2, and MSH6 in EGC.

Precision genome editing in plants via gene targeting and subsequent break-induced single-strand annealing.

Genome editing via artificial nucleases such as CRISPR/Cas9 has become popular in plants now. However, small insertions or deletions are major mutations and nucleotide substitutions rarely occur when DNA cleavage is induced. To induce nucleotide substitutions, a base editor utilizing dead or nickase-type Cas9 fused with deaminase have been developed.