Biology subjects

Mashimo, T.

Publications and source records attributed to Mashimo, T..

16 recordsLinked to original sources

bioRxiv2026

A rapidly deployable CRISPR-Cas3 diagnostic platform for emerging RNA viruses

Rapidly converting viral genome information into deployable molecular tests remains a major challenge in outbreak preparedness. We developed CONAN-SWIFT (Simple Workflow for Isothermal Field Testing), a sequence-to-test platform that integrates computational assay design, reverse-transcription loop-mediated isothermal amplification, CRISPR-Cas3 detection, reagent lyophilization and lateral-flow readout. Sequence-guided assays for Andes virus and Bundibugyo virus were established within approximately three weeks and extended to four additional filoviruses. A web-based designer supported crRNA selection, and systematic RT-LAMP primer optimization improved amplification performance. Recombinant Escherichia coli-expressed Cascade enabled standardized preparation of lyophilized Cas3-detection reagents, which were combined with a battery-operated isothermal device. The portable system detected as few as 10 input RNA copies per reaction within approximately 40 min. It also detected viral RNA and biologically contained, replication-incompetent Ebola virus in spiked human blood and concentrated wastewater. These findings establish the analytical feasibility of a rapidly adaptable CRISPR-Cas3 engineering framework for decentralized detection of emerging RNA viruses.

bioengineering↗

bioRxiv2026

A chorionic gonadotropin assay enables non-invasive detection of ovulation and early pregnancy in a New World primate model

Early detection of ovulation and pregnancy in the common marmoset is crucial for reproductive studies, yet hCG kits lack cross-reactivity with marmoset CG, and current methods remain labor-intensive. Here, we developed monoclonal antibodies against marmoset CG and CG{beta}, and established a non-invasive immunochromatographic CG assay. By eliminating invasive blood sampling, this assay supports 3Rs principles and enables practical endocrine monitoring. The assay detected urinary CG surges preceding ovulation, enabling efficient embryo recovery through artificial insemination (75%). Early pregnancy was detected at approximately 17 days post-ovulation. In addition, pregnancy detection in squirrel monkeys suggests conservation of CG features among certain New World primates. Overall, this simple, non-invasive assay provides a practical tool for marmoset research and establishes a foundation for future conservation-oriented reproductive monitoring following appropriate species-specific validation.

physiology↗

bioRxiv2025

A Novel MiRNA Detection System Combining TWJ-SDA,Multistep L-TEAM, and CRISPR-Cas3

MicroRNAs (miRNAs) serve critical regulatory roles in gene expression and are valuable biomarkers for early disease detection. However, their inherent low concentration in biological fluids poses significant detection challenges. Although traditional methods like real-time quantitative PCR (RT-qPCR) are highly sensitive, they require thermal cycling, limiting their application in point-of-care testing (POCT). Here, we present an isothermal amplification-based miRNA detection system integrating Three-Way Junction (TWJ) formation, Multistep Low-Temperature Amplification (L-TEAM), and CRISPR-Cas3-mediated signal amplification. The integration of the Multistep L-TEAM with the TWJ method achieves high sensitivity, detecting miRNA at concentrations as low as 10 femtomolar within 50 minutes, and effectively distinguishes single-nucleotide mismatches. When CRISPR-Cas3-mediated reaction was integrated, it still proved effective for confirming the presence of the target, but its quantitative reliability requires further optimization. We developed a predictive model using machine learning to facilitate rational optimization of experimental conditions through contribution analysis and to establish a methodology for designing more favorable sequences. The modular nature of our method permits adaptation to diverse miRNA targets without modifications to the fundamental amplification mechanism.

molecular biology↗

bioRxiv2025

Efficient gene disruption with CRISPR-Cas3 in human T cells

The CRISPR-Cas9 system has been widely adopted as a genome editing tool due to its high efficiency and versatility, contributing to the development of various therapeutic strategies. However, its clinical application remains limited by safety concerns, including off-target effects and large-scale chromosomal rearrangements such as translocations and inversions. Recently, the CRISPR-Cas3 system, a Class 1 CRISPR effector complex with unidirectional DNA degradation activity, has gained attention as a potential alternative, offering reduced off-target activity. In this study, we applied the CRISPR-Cas3 system to human T cells and successfully disrupted two clinically relevant genes, T cell receptor alpha constant (TRAC) and beta-2 microglobulin (B2M). These gene deletions were associated with a reduction in both graft-versus-host disease (GVHD) risk and host immune rejection. Importantly, no off-target mutations were detected in CRISPR-Cas3-edited cells, in contrast to the off-target effects observed with CRISPR-Cas9. Furthermore, CAR-T cells generated by deleting TRAC or B2M using CRISPR-Cas3 maintained their antigen-specific cytotoxicity against tumor cells, while exhibiting reduced alloreactivity. These results suggest that CRISPR-Cas3 provides a safer and promising platform for genome editing in T cell engineering, with potential applications in the development of next-generation allogeneic T cell therapies.

cancer biology↗

bioRxiv2025

Heritable Large Deletions Using Type I-E CRISPR-Cas3 in Rice

Type I-E CRISPR-Cas3 derived from Escherichia coli (Eco CRISPR-Cas3) can introduce large deletions in target sites and is available for mammalian genome editing. The use of Eco CRISPR-Cas3 to plants is challenging because 7 CRISPR-Cas3 components (6 Cas proteins and CRISPR RNA) must be expressed simultaneously in plant cells. To date, application has been limited to maize protoplasts, and no mutant plants have been produced. In this study, we developed a genome editing system in rice using Eco CRISPR-Cas3 via Agrobacterium-mediated transformation. Deletions in the target gene were detected in 39-48% and 55-71% of calli transformed with 2 binary vectors carrying 7 expression cassettes of Eco CRISPR-Cas3 components and a compact all-in-one vector carrying 3 expression cassettes of Cas proteins fused to 2A self-cleavage peptide, respectively. The frequency of alleles lacking a region 7.0 kb upstream of the PAM sequence was estimated as 21-61% by quantifying copy number by droplet digital PCR, suggesting that mutant plants could be obtained with reasonably high frequency. Deletions were determined in plants regenerated from transformed calli and stably inherited to the progenies. Sequencing analysis showed that deletions of 0.1-7.2 kb were obtained, as reported previously in mammals. Interestingly, deletions separated by intervening fragments or with short insertion and inversion were also determined, suggesting the creation of novel alleles. Overall, Eco CRISPR-Cas3 could be a promising genome editing tool for gene knockout, gene deletion, and genome rearrangement in plants.

plant biology↗

bioRxiv2023

Heart generation via blastocyst complementation in Mesp1/2-deficient mice

Heart transplantation is the only curative option available for patients with advanced heart failure. However, donor organ shortage and graft rejection remain critical challenges in heart transplantation. Blastocyst complementation using pluripotent stem cells (PSCs) can be used to generate organs such as the pancreas and kidneys in animal models with abnormalities in essential developmental genes. Nonetheless, whether functional adult hearts can be generated with blastocyst complementation remains unclear, and it is unknown if parenchyma, blood vessels, and stroma can be generated concomitantly from PSCs using blastocyst complementation to avoid graft rejection. Here, we show the generation of functional adult hearts in acardiac Mesp1 and Mesp2 double-knockout (Mesp1/2-DKO) mice via blastocyst complementation using mouse PSCs. Our result shows that the generated hearts were structurally and functionally normal and restored embryonic lethality in Mesp1/2-DKO mice. All four cardiovascular lineages, including cardiomyocytes, vascular endothelial cells, smooth muscle cells, and cardiac fibroblasts, were virtually entirely derived from exogenous PSCs in the myocardium. Exogenous rat PSCs also generated rat-derived xenogeneic hearts in Mesp1/2-DKO mice via interspecies blastocyst complementation. Thus, blastocyst complementation is a viable technique for generating hearts derived from PSCs and may represent significant progress toward generating rejection-free hearts.

cell biology↗

bioRxiv2023

G51D mutation of the endogenous rat Snca gene disrupts synaptic localisation of α-synuclein priming for Lewy-like pathology

Point mutations in the SNCA gene, encoding -synuclein (Syn), are a known cause of familial Parkinsons disease. The G51D mutation causes early onset neurodegeneration with complex pathology. We used CRISPR/Cas9 in rats to introduce the G51D mutation into the endogenous Snca gene. Co-localisation immunostaining studies with synaptic proteins showed that SynG51D protein is no longer efficiently localised to synapses. Furthermore, biochemical isolation of synaptosomes from rat cortex demonstrated a significant depletion of Syn in SncaG51D/+ and SncaG51D/G51D rats. Unbiased proteomic investigation of the cortex identified significant synaptic dysregulation in SncaG51D/G51D animals. Finally, we compared the propensity for Lewy-like pathology of Snca+/+ and SncaG51D/G51D rats by stereotaxically delivering Syn pre-formed fibrils (PFFs) into the pre-frontal cortex. At an early time-point, 6 weeks post-injection, we observed discrete Lewy-like structures positive for phosphoserine-129-Syn (pS129-Syn) only in SncaG51D/G51D brains. At 26 weeks post-injection of PFFs SncaG51D/G51D brains exhibited intense, discrete pS129-Syn-positive structures, while Snca+/+ brains exhibited diffuse pS129-Syn immunostaining. Quantification of discrete pS129-Syn-positive structures revealed the striatum of SncaG51D/G51D rats had significantly more Lewy-like pathology than Snca+/+ rats. In summary, this novel SncaG51D rat model exhibits molecular characteristics of early synaptic dysfunction and is primed for Syn pathology.

neuroscience↗

bioRxiv2023

A novel kit mutant rat enables hematopoietic stem cell engraftment without irradiation

Hematopoietic stem cell (HSC) transplantation is extensively studied in mouse models, but their limited scale presents challenges for effective engraftment and comprehensive evaluations. Rats, due to their larger size and anatomical similarity to humans, offer a promising alternative. In this study, we establish a rat model with the KitV834M mutation, mirroring KitW41 mice often used in KIT signaling and HSC research. KitV834M rats are viable and fertile, displaying anemia and mast cell depletion similar to KitW41 mice. The mutation affects myeloid cell proliferation and differentiation, as seen in the colony-forming unit granulocyte-macrophage assay. Importantly, KitV834M rats support donor rat-HSC engraftment without irradiation. Competitive transplantation assays reveal reduced reconstruction capacity in KitV834M HSCs. Leveraging the larger scale of this rat model enhances our understanding of HSC biology and transplantation dynamics, potentially advancing our knowledge in this field.

cell biology↗

bioRxiv2023

Txn1 mutation is a monogenic cause of chronic kidney disease associated with mitochondrial dysfunction in rats

Oxidative stress plays a significant role in the progression of chronic kidney disease. Thioredoxin 1 (Txn1) is one of the enzymatic antioxidants to regulate redox balance. However, the molecular mechanisms by Txn1 affects renal homeostasis remain unclear. This study aimed at elucidating the pathophysiology of Txn1 mutations in renal function. We used rats with the Txn1-F54L mutation generated by N-ethyl-N-nitrosourea mutagenesis. Laboratory tests and pathological examinations were performed in wild-type (WT) rats and in rats with heterozygous and homozygous Txn1-F54L mutations. We performed RNA-seq analysis of the WT and homozygotes. To confirm phenotypic reproducibility, the Txn1-F54L mutation was generated in rats with different genetic backgrounds using CRISPR/Cas9 genome editing technology. Txn1-F54L mutant rats exhibited progressive albuminuria, hypoalbuminemia, hypercholesterolemia, and hypertension. Renal pathology revealed marked nephrosclerosis, tubular dilatation, interstitial fibrosis, and decreased number of mitochondria, mainly in the paroxysmal tubules. We confirmed a similar phenotype of chronic kidney disease (CKD) in different rat strains. RNA-seq showed the downregulation of mitochondria-related genes and significant upregulation of genes associated with inflammation, pyroptosis, apoptosis, and necroptosis in mutant rats. Our results show that the Txn1 mutation is a monogenic cause of CKD termination. The underlying pathology involves several regulated cell-death pathways. Thus, our study provides a new animal model of oxidative stress-induced CKD. Txn1-F54L mutant rats will aid in developing therapeutic strategies for CKD. Translational StatementWe found that the deficiency of thioredoxin (Txn1), which regulates oxidative stress, spontaneously caused chronic kidney disease (CKD) in rats. The Txn1-F54L (Adem) rat is a new model of CKD with complications such as anemia, hypertension, and cardiovascular disease. Renal pathology revealed nephrosclerosis, interstitial fibrosis, and mitochondrial damage. The molecular basis of the underlying pathologies included inflammation, pyroptosis, apoptosis, and necroptosis. These pathological changes are partially linked to renal diseases such as diabetic nephropathy, hypertensive nephrosclerosis, and ischemic reperfusion injury. Adem rats could help understand the common pathological mechanisms of these renal diseases and develop therapeutic strategies.

physiology↗

bioRxiv2023

Opioidergic activation of descending pain inhibitory system underlies placebo analgesia

Placebo analgesia is caused by inactive treatment, implicating endogenous brain function involvement. However, the underlying neurobiological mechanisms remain unclear. We found that -opioid signals in the medial prefrontal cortex (mPFC) activate the descending pain inhibitory system to initiate placebo analgesia in neuropathic pain rats. Chemogenetic manipulation demonstrated that specific activation of -opioid receptor-positive (MOR+) neurons in the mPFC or suppression of the mPFC-ventrolateral periaqueductal gray (vlPAG) circuit inhibited placebo analgesia in rats. MOR+ neurons in the mPFC are monosynaptically connected and directly inhibit L5 pyramidal neurons that project to the vlPAG via GABAA receptors. Thus, intrinsic opioid signaling in the mPFC disinhibits excitatory outflow to the vlPAG by suppressing MOR+ neurons, leading to descending pain inhibitory system activation that initiates placebo analgesia. One Sentence SummarySugar pills relieve pain by activating the intrinsic pain inhibitory system via opioidergic signals in the prefrontal cortex.

neuroscience↗

bioRxiv2022

In vivo 18F-DOPA PET imaging identifies a dopaminergic deficit in a rat model with a G51D α-synuclein mutation

Parkinsons disease (PD) is a neurodegenerative condition with several major hallmarks, including loss of substantia nigra neurons, reduction in striatal dopaminergic function, and formation of -synuclein-rich Lewy bodies. Mutations in SNCA, encoding for -synuclein, are a known cause of familial PD, and the G51D mutation causes a particularly aggressive form of the condition. CRISPR/Cas9 technology was used to introduce the G51D mutation into the endogenous rat SNCA gene. SNCAG51D/+ and SNCAG51D/G51D rats were born in Mendelian ratios and did not exhibit any severe behavourial defects. L-3,4-dihydroxy-6-18F-fluorophenylalanine (18F-DOPA) positron emission tomography (PET) imaging was used to investigate this novel rat model. Wild-type (WT), SNCAG51D/+ and SNCAG51D/G51D rats were characterised over the course of ageing (5, 11, and 16 months old) using 18F-DOPA PET imaging and kinetic modelling. We measured the influx rate constant (Ki) and effective distribution volume ratio (EDVR) of 18F-DOPA in the striatum relative to the cerebellum in WT, SNCAG51D/+ and SNCAG51D/G51D rats. A significant reduction in EDVR was observed in SNCAG51D/G51D rats at 16 months of age indicative of increased dopamine turnover. Furthermore, we observed a significant asymmetry in EDVR between the left and right striatum in aged SNCAG51D/G51D rats. The increased and asymmetric dopamine turnover observed in the striatum of aged SNCAG51D/G51D rats is similar to prodromal PD, which suggests the presence of compensatory mechanisms. SNCAG51D rats represent a novel genetic model of PD, and kinetic modelling of 18F-DOPA PET data has identified a highly relevant early disease phenotype.

neuroscience↗

bioRxiv2022

Catecholaminergic cell type-specific expression of Cre recombinase in knock-in transgenic rats generated by the Combi-CRISPR technology

BackgroundCell groups containing catecholamines provide a useful model to study the molecular and cellular mechanisms underlying the morphogenesis, physiology, and pathology of the central nervous system. For this purpose, it is necessary to establish a system to induce catecholaminergic group-specific expression of Cre recombinase. Recently, we introduced a gene cassette encoding 2A peptide fused to Cre recombinase into the site between the C-terminus and translational termination codons of the rat tyrosine hydroxylase (TH) open reading frame by the Combi-CRISPR technology, which is a genomic editing method to enable an efficient knock-in (KI) of long DNA sequence into a target site. However, the expression patterns of the transgene and its function as well as the effect of the mutation on the biochemical and behavioral phenotypes in the KI strains have not been characterized yet. New MethodWe aimed to evaluate the usefulness of TH-Cre KI rats as an experimental model for investigating the structure and function of catecholaminergic neurons in the brain. ResultsWe detected cell type-specific expression of Cre recombinase and site-specific recombination activity in the representative catecholaminergic groups in the TH-Cre KI rat strains. In addition, we measured TH expression level and catecholamine accumulation in the brain regions, and spontaneous locomotion, indicating that catecholamine metabolism and general behavior are apparently normal in these KI rats. ConclusionsTH-Cre KI rat strains produced by the Combi-CRISPR system offer a beneficial model to study the molecular and cellular mechanics for the morphogenesis, physiology, and pathology of catecholamine-containing neurons in the brain.

neuroscience↗

bioRxiv2022

Highly selective transgene expression through double-floxed inverted orientation system by using a unilateral spacer sequence

The double-floxed inverted orientation (DIO) system with adeno-associated viral (AAV) vector provides a beneficial approach to express transgenes in specific cell populations having Cre recombinase. A significant issue with this system is the protection against non-specific expression of transgenes in tissues after vector injection. We here show that Cre-independent recombination in AAV genome carrying the DIO sequence occurs during the production of viral vectors in packaging cells, which results in transgene expression in off-target populations. Introduction of a relatively longer nucleotide sequence between two recognition sites at the unilateral side of the transgene cassette, termed a unilateral spacer sequence (USS), is useful to suppress recombination during the vector production, leading to the protection of non-specific transgene expression with enhanced gene expression selectivity. Our DIO/USS system offers a powerful strategy for highly specific Cre-dependent transgene expression, aiming at various applications for structural and functional analyses of target cell populations.

neuroscience↗

bioRxiv2021

Txn1 mutation causes epilepsy associated with vacuolar degeneration in the midbrain

Thioredoxin (TXN), encoded by Txn1, acts as a critical antioxidant in the defense against oxidative stress by regulating the dithiol/disulfide balance of interacting proteins. The role of TXN in the central nervous system (CNS) is largely unknown. A phenotype-driven study of N-ethyl-N-nitrosourea-mutated rats with running seizures at around five-week of age revealed the relevance of Txn1 mutations to CNS disorders. Genetic mapping identified Txn1-F54L in epileptic rats. The insulin-reducing activity of Txn1-F54L rats was approximately one-third that of the wild-type. Vacuolar degeneration in the midbrain, mainly in the thalamus and the inferior colliculus, was observed in the Txn1-F54L rats. The lesions displayed neuronal and oligodendrocyte cell death. Neurons in Txn1-F54L rats showed morphological changes in the mitochondria. Vacuolar degeneration began at three weeks of age, and spontaneous repair began at seven weeks; a dramatic change from cell death to repair occurred in the midbrain during a restricted period. In conclusion, Txn1 is essential for the development of the midbrain in juvenile rats.

neuroscience↗

bioRxiv2021

Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific ssDNA cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target dsDNA substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

molecular biology↗

bioRxiv2020

ASCL1 regulates neurodevelopmental transcription factors and cell cycle genes in glioblastoma

Glioblastomas (GBMs) are incurable brain tumors with a high degree of cellular heterogeneity and genetic mutations. Transcription factors that normally regulate neural progenitors and glial development are aberrantly co-expressed in GBM, conferring cancer stem-like properties to drive tumor progression and therapeutic resistance. However, the functional role of individual transcription factors in GBMs in vivo remains elusive. Here, we demonstrate that the basic-helix-loop-helix (bHLH) transcription factor ASCL1 regulates transcriptional targets that are central to GBM development, including neural stem cell and glial transcription factors, oncogenic signaling molecules, chromatin modifying genes, and cell cycle and mitotic genes. We also show that the loss of ASCL1 significantly reduces the proliferation of GBMs induced in the brain of a genetically relevant glioma mouse model, resulting in extended survival times. RNA-seq analysis of mouse GBM tumors reveal that the loss of ASCL1 is associated with downregulation of cell cycle genes, illustrating an important role for ASCL1 in controlling the proliferation of GBM. TABLE OF CONTENTSO_ST_ABSMain PointsC_ST_ABSO_LIASCL1 is co-expressed with neural stem cell/glial transcription factors in GBM C_LIO_LIASCL1 binds to genes that are important for cell proliferation and cancer in the brain. C_LIO_LILoss of ASCL1 downregulates cell cycle genes and increase survival of glioma mouse model. C_LI Table of Content Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/958132v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@8aa2d7org.highwire.dtl.DTLVardef@1c908edorg.highwire.dtl.DTLVardef@1690085org.highwire.dtl.DTLVardef@a13728_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗