Yasuhiro Kazuki

Background/Objectives: Mammalian display enables direct screening of full-length IgG antibodies, but genetically controlled library construction remains technically demanding. Here, we developed a mammalian display platform in which a target-naïve ΔDH antibody scaffold is first established at a defined human artificial chromosome (HAC) acceptor site, and CDR-H3 diversity is then introduced directly into that fixed genomic background by CRISPR-Cas9-mediated homology-directed repair (HDR). Methods: A site-specifically integrated display cell line carrying the ΔDH scaffold on an HAC was generated by Cre-mediated recombination. Randomized CDR-H3 libraries were introduced into this defined genomic background by HDR and screened by magnetic- and fluorescence-activated cell sorting together with orthogonal antigen-detection formats. Binding of isolated clones was characterized by flow cytometry and enzyme-linked immunosorbent assay. Results: Although early selection enriched clones reactive with a fluorescent reagent rather than the intended antigen, refinement of the selection scheme enabled isolation of AFFVVF. This selected antibody variant recognized multiple SARS-CoV-2 receptor-binding domain (RBD) variants by flow cytometry, with no detectable binding to unrelated antigens. As a recombinant protein, AFFVVF bound the JN.1 and XEC spike trimers by ELISA, but not monomeric RBD, including the XEC RBD used for selection, indicating that the form of antigen recognized differed between the cell-based and plate-based assays. Exploratory selections against epidermal growth factor receptor (EGFR) and glycoprotein A33 (GPA33) also produced antigen-dependent population shifts and enrichment of discrete sequence variants, although individual candidates were not independently validated as recombinant antibodies. Conclusions: These results provide proof of principle that targeted CDR-H3 diversification within a pre-established, genetically defined, target-naïve full-length IgG scaffold can yield a new antigen-binding antibody without starting from a pre-existing antigen-specific antibody or repertoire.

Fear conditioning is widely used to assess associative memory in mice, yet percent freezing conflates memory with baseline locomotor and anxiety-related traits. A systematic survey of recent studies (2020–2025) found that fewer than 1% statistically integrate locomotor activity into freezing analyses. Here, we address this gap using a large-scale dataset of >10,000 mice across >160 comparisons, including genetic mutations, pharmacological interventions and aging, tested in 15 standardized behavioral paradigms. Conventional freezing scores covaried strongly with general locomotor activity, obscuring memory-related phenotypes. Multiple factor analysis identified two principal behavioral dimensions, locomotor activity and learning/memory: conventional freezing aligned with the locomotor dimension, whereas freezing subtraction and the activity suppression ratio mapped onto the memory dimension and improved detection of synaptic plasticity phenotypes. These analyses show that baseline locomotor normalization is essential for interpreting fear conditioning as a memory assay and provide an open framework for selecting and reporting locomotor-normalized metrics.

Centromeres are specialized chromatin structures essential for equal chromosome segregation. The human centromeres are organized on a portion of homogeneously repeated DNA sequence and contains nucleosomes including centromere specific histone H3 variant CENP-A. Histone modifications around the CENP-A dense region are also thought to have important roles in centromere function. Here, we used human artificial chromosome (HAC) system based on synthetic centromeric repeat DNA to enable structural and chromatin analysis at ∼2-kb resolution within defined centromere core domains. Analysis using this HAC centromere showed that the CENP-A dense regions span approximately 18-50 kb in size and coexisted with euchromatic histone modifications in an interdependent manner. We further found that when DNA replication reduced CENP-A density, a heterochromatin modification H3K9me3 transiently accumulated in the CENP-A-dense regions. This accumulation was suppressed by the CENP-A deposition factor HJURP and the histone acetyltransferase KAT7, suggesting that CENP-A assembly and euchromatic modifications interdependently antagonize heterochromatin accumulation. The synthetic centromere DNA generated in this study elucidates the epigenetic landscape within the centromere core regions and provides a more precise framework for understanding the dynamic balance between CENP-A assembly and histone modifications.

Traditional Japanese medicines (Kampo medicines) are increasingly used concurrently with other medications, but evidence regarding their potential for drug-drug interactions (DDIs) is insufficient. Given the important role of cytochrome P450 3A (CYP3A) in DDIs, this study assessed the CYP3A-mediated DDI risk of three Kampo medicines (Hochuekkito, Goreisan and Ninjin'yoeito).We utilised a CYP3A-humanised mouse model to comprehensively evaluate the effects of the three Kampo medicines on CYP3A activity, including an analysis of the area under the plasma concentration-time curve (AUC) and AUC ratio (AUCR).None of the three Kampo medicines tested significantly affected the AUC of the CYP3A substrate triazolam or the AUCRs of triazolam metabolites.4. These results provide important preclinical evidence indicating the lack of a significant net effect on CYP3A-mediated DDIs for Hochuekkito, Goreisan and Ninjin'yoeito under the tested repeated-dosing conditions, thereby supporting their safe and proper use.

Quantifying low-frequency chromosomal alterations in living cell populations at early stages is essential in many fields including cancer studies and chromosome engineering, yet selection-based readouts impose delays and can lose fragile positives before readout, biasing frequency estimates; CRISPR imaging rarely reports detection limits at 10^−4. Here, we developed High-throughput CRISPR Imaging (Hi-CRI), integrating engineered dCas9-sgRNA ribonucleoprotein (RNP) labeling, suppression of nonspecific aggregates via metabolic modulation and protease treatment, high-speed volumetric imaging by oblique plane microscopy, GPU-accelerated image analysis, and an explicit error-controlled detection-limit framework. Using per-cell signal-to-noise ratio calling, Hi-CRI achieves a 0.01% detection limit for target-positive cell fractions. In microcell-mediated chromosome transfer of a mouse artificial chromosome (MAC) into HT1080 recipients, Hi-CRI measured 0.03% MAC-positive cells among 184,235 recipients at day 1 post-fusion, versus 0.0007% by antibiotic-selection-based clonogenic assay at day 8 post-fusion, consistent with substantial loss before readout (pre-readout attrition). Hi-CRI enables viability-preserving, selection-independent quantification of low-frequency chromosomal states.

Mesenchymal stromal cells (MSCs) are widely used in regenerative medicine, but their clinical utility is limited by replicative senescence. Strategies that

Human/mouse artificial chromosomes (HACs/MACs) serve as gene vectors for transferring of megabase DNA sequences and multiple gene cassettes into mammalian cells. However, their full potential is hindered by the lack of effective gene-loading methods. In this study, we developed a fragment assembly (FA) system that enables reconstitution of introduced sequences in HACs/MACs by simultaneously loading multiple gene-loading vectors (GLVs) while removing the GLV vector backbone sequences. As a proof of principle, we simultaneously loaded three GLVs, which encode the promoter, open reading frame of a gene, and a poly A signal sequence, onto a MAC carrying a gene-loading pad for the FA system. We successfully obtained clones bearing a reconstituted gene expression cassette on the MAC. Further introduction of three GLVs in two gene-loading steps enabled the reconstitution of another gene expression cassette in the MAC. The gene-loaded MAC was transferable to other cells, and the introduced cells expressed the reconstituted genes. These data demonstrate that the FA system is useful for reconstituting multiple sequences in HACs/MACs.

Humanized transgenic mice carrying human genes are useful for research on gene function and disease. Bacterial artificial chromosomes (BACs) that carry human genomic sequences with regulatory elements enable the expression of transgenes at physiological levels in vivo. To study complex biological phenomena involving multiple genes, techniques for co-introducing transgenes into mice have been developed; however, the introduction of multiple BACs remains laborious. The simultaneous integration of multiple gene loading vectors (SIM) system was developed to incorporate three or more gene-loading vectors (GLVs) using a mouse artificial chromosome (MAC) vector. This system allows for simultaneous site-specific incorporation of three GLVs into a single MAC with only one screening. However, the capacity for large constructs, such as BACs, has yet to be evaluated. This study is the first to demonstrate the development of multi-BAC transchromosomic (Tc) mice targeting the human leukocyte antigen (HLA) class I gene cluster (HLA-A, HLA-B, HLA-C) and beta-2-microglobulin (B2M) using the SIM system. By constructing a MAC using three BACs containing these genomic regions, we successfully generated HLA class I Tc mice. The technology to generate multi-BAC Tc mice will accelerate the analysis of complex life mechanisms involving multiple factors.

The human cytochrome P450 3A4 (CYP3A4) enzyme, which is expressed in the liver and the intestines, catalyzes the metabolism of numerous drugs. The induction of CYP3A4 by the pregnane X receptor (PXR) represents a significant problem in pharmacotherapy. Since the metabolic properties of CYP3A substrates and the recognition of PXR ligands differ between humans and rats, there are limitations to reproducing human CYP3A4 induction in rat studies. Here, we developed a double-humanized rat model for CYP3A and PXR to mimic drug-drug interactions in humans using a rat model. The model was created using a combination of mammalian artificial chromosome-based chromosome engineering, bacterial artificial chromosome-based transgenic technology, and genome editing. The resulting double-humanized rat model for CYP3A and PXR (huCYP3A-PXR rat) was treated with rifampicin, a human PXR ligand. This treatment increased CYP3A4 mRNA expression in the liver and intestine and enhanced the hydroxylation activities of triazolam, a CYP3A4 substrate. Pretreatment with rifampicin resulted in a reduction of triazolam plasma concentrations and an increase in its metabolites after oral administration. Furthermore, ketoconazole, a CYP3A4 inhibitor, led to an increase in triazolam plasma concentrations and a decrease in its metabolites in huCYP3A-PXR rats regardless of pretreatment with rifampicin. These results suggest that huCYP3A-PXR rats could predict human CYP3A4 induction and inhibition in vivo. This innovative model, in which PXR and CYP3A are humanized, is expected to be a valuable tool for studying drug-drug interactions, including a combination of inducers and inhibitors, and to be integrated into the preclinical drug-development pipeline in future.