Biology subjects

Igawa, T.

Publications and source records attributed to Igawa, T..

8 recordsLinked to original sources

bioRxiv2026

Generation of lachrymatory factor synthase-suppressed onion (Allium cepa L.) by Agrobacterium-mediated gene transfer for CRISPR/Cas9 genome editing

Lachrymatory factor, an irritating volatile with tear-inducing property, is produced when onion bulbs are cut or chopped. We aimed to generate onion plants with reduced lachrymatory factor synthase (LFS) activity via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) genome editing. Calli induced from primary roots were transformed with Agrobacterium tumefaciens carrying expression cassettes for CRISPR/Cas9, guide RNA, green fluorescent protein (GFP), and hygromycin resistance; callus lines that showed a high-frequency stable GFP expression were selected as "elite callus lines" that were suitable for transformation. Cleaved amplified polymorphic sequence (CAPS), heteroduplex mobility assay (HMA), and Sanger sequencing confirmed mutations introduced into the LFS gene, and plants were regenerated from the confirmed LFS-edited callus lines. The LFS enzyme activity in the leaves and bulbs of the LFS-edited plants was lower than that in control plants, while the LFS-edited plants exhibited severe growth abnormalities and failed to set seed, possibly due to long-term culture to maintain the elite callus line. The present study first demonstrated that onion genome editing, which modified a specific trait of onion, the reduction of LFS activity, was achieved. The results obtained opened the feasible way toward the final goal: the production of tear-free, higher health-functional onions.

plant biology↗

bioRxiv2026

Transcriptome-based genome-wide analysis reveals hybridization dynamics and genetic structure of Japanese giant salamanders

AbstractsThe Japanese giant salamander (Andrias japonicus), an apex predator and a Special Natural Monument in Japan, is threatened by hybridization with introduced Chinese giant salamanders (Andrias davidianus). This hybridization has caused genetic introgression and expansion of hybrid populations, posing a serious conservation risk. Because morphological identification of hybrids is occasionally unreliable and current genetic methods rely on limited markers, a genome-wide approach is required. However, the extremely large genome ([~]50 Gb) of giant salamanders has hindered whole-genome analyses. In this study, we conducted transcriptome-based analyses of Japanese giant salamanders, Chinese giant salamanders, and their hybrids, generating RNA-seq data from 34 individuals. A total of over 419,000 SNP candidates were identified, from which 4,457 high-confidence SNPs in highly expressed genes were selected for analysis. Population structure analyses for Nabari colony revealed that hybrid individuals form two major groups, corresponding to different degrees of genetic contribution from Japanese and Chinese lineages. Most hybrids were inferred to be F2 or backcross individuals, while F1 hybrids were rare. Mitochondrial analysis indicated that all hybrids possessed Japanese-type mitochondrial genome, suggesting male-mediated introgression from Chinese salamanders. Differential expression analysis revealed enhanced stress-response pathways in hybrids and stronger antiviral responses in Japanese individuals. Using the axolotl genome as a reference, we constructed a virtual chromosomal map, identifying large haplotype blocks and supporting recent hybridization with limited recombination. This study provides a genome-wide framework for understanding hybridization dynamics and supports future conservation and evolutionary studies.

ecology↗

bioRxiv2025

Whole-genome sequencing of CRFK and PG-4 cells to infer the phenotype of the original donors

BackgroundCrandell-Rees Feline Kidney (CRFK; kidney-derived) cells and PG-4 cells (astrocyte-derived) have been in use and have been passaged for decades in laboratories worldwide; however, no detailed information on the genetic background of the donor individuals is available, particularly regarding phenotype characteristics such as coat and iris color. ResultsWe performed whole-genome sequencing of CRFK and PG-4 cells. We analyzed the resulting data to infer the phenotype of the individual from which the cells were derived, specifically for the coat color, coat length, coat pattern, and iris color. Our data suggested that CRFK cells originated from a cat with long black fur lacking stripes and with non-blue irises; PG-4 cells originated from a cat with long bicolored white and black fur without stripes, and with non-blue irises. Analysis of publicly available RNA-seq data confirmed that genes associated with coat phenotype and iris color are expressed in the skin and eyes, as well as in various other organs. ConclusionsVariants of the genes affecting coat phenotype and iris color may influence physiological functions throughout the body. These results shed light on the previously unknown genetic background of commonly used feline cultured cells and the phenotype of the donor individuals. These findings may facilitate more accurate interpretation of data obtained from cultured feline cells and provide guidelines for developing cell lines with domestic cat genotypes exhibiting diverse phenotypes through genome editing. This will help to provide clarity regarding deafness in cats with white fur and blue irises and elucidate the effects of melanocyte destruction caused by KIT gene mutations on the nervous system and the influence of other coat-related factors in organs other than the skin or functions independent of coat formation.

genomics↗

bioRxiv2025

Comparative Genomic Analysis of Cyanobacteria as Amphibian Food Sources: Insights into High-Temperature Tolerance Potential

Hot-springs represent one of the most accessible yet extreme environments on the Earths surface. We isolated and characterized two novel cyanobacterial strains of the genus Leptolyngbya, L. sp. Akita and L. sp. Seranma from geothermal hot-springs in Japan. These strains showed distinct morphological features; L. sp. Akita exhibited linear filamentous structures and blue-green color when cultured under white light, whereas L. sp. Seranma exhibited loosely coiled structures and a brown color under the same conditions. Whole-genome sequencing and comprehensive genomic analyses of their genomes identified the acquisition of gene clusters related to metabolic processes, which may be associated with strain-specific phenotypes. In addition, intestinal metagenomic analysis of tadpoles (Buergeria buergeri and B. japonica) sympatrically living with these strains in the hot-springs suggested that the tadpoles utilized these Leptolyngbya as temporal or regular foods in high-temperature environments. These findings provide insights into the ecological significance of these cyanobacteria in extreme environments and their potential applications in biotechnology and the ecological conservation of primary consumers, including amphibians.

microbiology↗

bioRxiv2025

Production of homozygous deletion mutants targeting fertilization regulator genes through multiplex genome editing

Producing the gene-knockout mutant is a critical strategy in reverse genetics for gene functional analyses. Plant science has applied various mutagenesis, including chemical mutagen, T-DNA insertion, and genome editing. The intact gene expression is completely disrupted, while the mutant retains the intact sequence region. Lately, effective whole open reading frame (ORF) deletion through the CRISPR/Cas9 system using multiplex guide RNAs was reported in Arabidopsis, targeting a gene expressed in somatic cells. Here we applied the scheme targeting the reproductive genes GENERATIVE CELL SPECIFIC 1(GCS1), GAMETE EXPRESSED 2 (GEX2), DUF679 DOMAIN MEMBRANE PROTEIN 8 (DMP8), and DMP9, which are fertilization regulators. Homozygous gene deletion lines were successfully obtained in the T1 generation, with acquisition rates ranging from 8.3% to 30.0%. The rates approximately correlated with the scores predicted by the DeepSpCas9. The analysis of the GCS1 deletion line ({Delta}gcs1) suggested that avoiding the first bolt removal is important for consistent phenotype of the developed inflorescences. Compared to the previously reported mutants, the GEX2 deletion line ({Delta}gex2) showed a difference in seed development phenotype, indicating that the remaining intact gene region in the mutant could unexpectedly influence the function. Simultaneous deletions of DMP8 and DMP9, which are in distinct chromosomes, were also succeeded in the T1 generation. The obtained results showed that all deletions were inheritable. The scheme challenged here demonstrated the effective production of homozygous mutants for the genes, even if the reproductive lethal or recessive.

plant biology↗

bioRxiv2022

Rapid and collective determination of the complete "hot-spring frog" mitochondrial genome containing long repeat regions using Nanopore sequencing

The mitochondrial genome (mt-genome) is one of the promising molecular markers for phylogenetics and population genetics. Recently, various mt-genomes have been determined rapidly by using massively parallel sequencers. However, the control region (CR, also called D-loop) in mt-genomes remain difficult to precisely determine due to the presence of repeat regions. Here, using Nanopore sequencing, we succeeded in rapid and collective determination of complete mt-genome of the hot-spring frog, Buergeria japonica, and found that its mt-genome size was 22,274 bp including CR (6,929 bp) with two types of tandem repeat motifs forming repeat regions. Comparison of assemble strategies revealed that the long- and short-read data combined together enabled efficient determination of the CR, but the short-read data alone did not. The B. japonica CR was longer than that of a congenic species inhabiting cooler climate areas, Buergeria buergeri, because of the long repeat regions in the former. During the thermal adaptation of B. japonica, the longer repeat regions in its CR may have accumulated within a short period after divergence from B. buergeri.

genomics↗

bioRxiv2022

Molecular engineering of emicizumab to improve hemophilia A treatment

Emicizumab, a factor (F)VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment for people with hemophilia A (PwHA). Although emicizumab is very potent, long-term outcomes from the clinical studies suggest that a small proportion of PwHA still experiences bleeds. Additionally, non-clinical studies indicate that the maximum cofactor activity of emicizumab is lower than international standard activity (100 IU/dL of FVIII). An increased cofactor activity BsAb would benefit such patients. Here, we report NXT007, a BsAb binding FIXa and FX developed through further engineering of emicizumab. Emicizumab has a common light chain, but through advances in antibody engineering, we were able to create a more potent BsAb with two new non-common light chains. After extensive optimization of the heavy and light chains, the resulting BsAb, NXT007, exerted in vitro thrombin generation (TG) activity in hemophilia A plasma equivalent to 100 IU/dL of FVIII when triggered by tissue factor. NXT007 demonstrated potent hemostatic activity in an acquired hemophilia A model in non-human primates at a much lower dosage than emicizumab, consistent with an around 30-fold dose shift in the in vitro TG activity between NXT007 and emicizumab. Moreover, together with Fc engineering that enhanced FcRn binding and reduced in vivo clearance, we demonstrate that NXT007 could be effective at a much lower dosage with a longer dosing interval compared to emicizumab. These non-clinical results suggest that NXT007 could maintain a non-hemophilic range of coagulation potential in PwHA and provides a rationale for its clinical testing.

bioengineering↗

bioRxiv2017

The widespread misconception about the Japanese major biogeographic boundary, the Watase line (Tokara gap), revealed by bibliographic and beta diversity analyses

The biota of the Japanese Archipelago is divided into the Palearctic and Oriental realms by the Watase line (Tokara gap), a major biogeographic boundary of Japan. This boundary is generally placed between Akusekijima and Kodakarajima Islands of the Tokara Archipelago, and has been the subject of many biogeographic debates. However, despite being widely accepted, the position of the boundary is doubtful because of a lack of clear evidence. Here, to verify the definition and existence of the biogeographic boundary, we performed a documentary search and beta diversity analysis of multiple taxa. Our documentary search suggested that the Watase line (Tokara gap) should be put between Yakushima/Tanegashima and Amamioshima Islands, but recent references to it clearly deviate from its original definition, and that the placement of the boundary line between Akusekijima and Kodakarajima Islands is based on limited and biased evidence. Our beta diversity analyses found no common biogeographic boundary dividing the Tokara Archipelago into two realms, and showed that the beta diversity pattern of this region is explained by the areas and geographic distances of the islands in agreement with the general principles of island biogeography. The widespread misunderstanding of biogeography in this region could have been perpetuated by preconception and the citation of references without verification. Our study proposes that revision of the biogeography in the Tokara Archipelago, a gap region between the Palearctic and Oriental realms, is necessary and demonstrates the negative influence of preconception in biogeographic debate.

ecology↗