Biology subjects

Miyaoka, Y.

Publications and source records attributed to Miyaoka, Y..

10 recordsLinked to original sources

bioRxiv2026

Multi-color droplet digital PCR assay enables allele-specific quantification of heterogeneous genome editing outcomes

Precise characterization of genome editing outcomes remains a major challenge because edited cell populations contain diverse alleles generated by homology-directed repair, non-homologous end joining (NHEJ), or base editing. While next-generation sequencing enables comprehensive analysis, its routine use is constrained by cost and turnaround time. Here, we developed a multi-color droplet digital PCR (ddPCR) assay that exploits six-color fluorescence detection to quantitatively distinguish multiple edited alleles within a single reaction. Using CRISPR-Cas9 and base editing model systems, we designed sequence-specific probe sets that distinguished recurrent NHEJ alleles generated by CRISPR-Cas9 editing as well as target and bystander alleles generated by base editing. The assay quantitatively resolved individual editing outcomes that could not be distinguished by conventional Sanger sequencing. Together, these results establish multi-color ddPCR as a rapid, scalable, and sequence-specific approach for quantification of genome editing outcomes across multiple editing modalities.

molecular biology↗

bioRxiv2026

High-throughput CRISPR live-cell imaging of low-frequency chromosomal events quantifies the latent efficiency of chromosome engineering

Abstract/SummaryQuantifying 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. TeaserRare chromosome events can be counted in living cells by high-throughput CRISPR imaging before selection hides them.

bioengineering↗

bioRxiv2025

Derivation of human post-mitotic cardiomyocytes from tetraploid iPSCs

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPS-CMs) have great potential in regenerative medicine. However, iPS-CMs are immature and resemble fetal cardiomyocytes, restricting their application. Although fetal cardiomyocytes in the human heart lose their proliferative potential during maturation and become tetraploid, iPS-CMs cannot replicate this tetraploidization and remain immature. To overcome this problem, we fused diploid iPSCs to establish tetraploid iPSCs and differentiated them into cardiomyocytes (4N-iPS-CMs). We found that 4N-iPS-CMs had more similar gene expression profiles, mitochondrial amounts, contractile impedance, and resistance to a potassium blocker in post-mitotic cardiomyocytes than conventional iPS-CMs. In addition, we successfully generated 4N-iPS-CMs from two individuals to mix two different genetic backgrounds. Thus, we demonstrated a novel strategy for generating human post-mitotic cardiomyocyte-like cells by generating tetraploid iPSCs.

cell biology↗

bioRxiv2024

High-throughput robotic isolation of human iPS cell clones reveals frequent homozygous induction of identical genetic manipulations by CRISPR-Cas9

Genome editing in human iPS cells is a powerful approach in regenerative medicine. CRISPR-Cas9 is the most common genome editing tool, but it often induces byproduct insertions and deletions in addition to the desired edits. Therefore, genome editing of iPS cells produces diverse genotypes. Existing assays mostly analyze genome editing results in cell populations, but not in single cells. However, systematic profiling of genome editing outcomes in single iPS cells was lacking. In this study, we developed a method for high-throughput iPS cell clone isolation based on the precise robotic picking of cell clumps derived from single cells grown in extracellular matrices. We analyzed over 1,000 genome-edited iPS cell clones and found that homozygous editing was much more frequent than heterozygous editing. We also observed frequent homozygous induction of identical genetic manipulations, including insertions and deletions. Our new cloning method and findings will facilitate the application of genome editing to human iPS cells.

bioengineering↗

bioRxiv2023

Fusion of histone variants to Cas9 enhances homology-directed repair

As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 exhibited the improved HDR/NHEJ ratio better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing.

cell biology↗

bioRxiv2023

In Vivo Delivery of Therapeutic Molecules by Transplantation of Genome-Edited Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (iPSCs) have already been used in transplantation therapies. Currently, cells from healthy people are transplanted into patients with diseases. With the rapid evolution of genome editing technology, genetic modification could be applied to enhance the therapeutic effects of iPSCs, such as the introduction of secreted molecules to make the cells a drug delivery system. Here, we addressed this possibility by utilizing a Fabry disease mouse model, as a proof of concept. Fabry disease is caused by the lack of -Galactosidase A (GLA). We previously developed an immunotolerant therapeutic molecule, modified -N-acetylgalactosaminidase (mNAGA). We confirmed that secreted mNAGA from genome-edited iPSCs compensated for the GLA activity in GLA-deficient cells using an in vitro co-culture system. Moreover, iPSCs transplanted into Fabry model mice secreted mNAGA and supplied GLA activity to the liver. This study demonstrates the great potential of genome-edited iPSCs secreting therapeutic molecules.

cell biology↗

bioRxiv2022

Granulin Loss of Function in Human Mature Brain Organoids Implicates Astrocytes in TDP-43 Pathology

Loss of function (LoF) of Tar-binding protein 43 (TDP-43) and mislocalization, together with TDP-43-positive and hyperphosphorylated inclusions, are found in postmortem tissue of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those carrying LoF variants in the progranulin gene (GRN). Modelling TDP-43 pathology has been challenging in vivo and in vitro. We present a 3D-induced pluripotent stem cell (iPSC)-derived paradigm - mature brain organoids (mbOrg) - composed of cortical-like-astrocytes (iA) and neurons (iN). When devoid of GRN, mbOrgs spontaneously recapitulate TDP-43 mislocalization, hyperphosphorylation and LoF phenotypes. Mixing-and-matching genotypes in mbOrgs showed that GRN-/- iA are drivers for TDP-43 pathology. Finally, we rescued TDP-43 LoF by adding exogenous progranulin, demonstrating a link between TDP-43 LoF and progranulin expression. In conclusion, we present an iPSC-derived platform that shows striking features of human TDP-43 proteinopathy and provides a tool for mechanistic modelling of TDP-43 pathology and patient-tailored therapeutic screening for FTD and ALS. HighlightsO_LIGRN-/- iPSC-derived 3D paradigm (mbOrg) composed of mature cortical-like astrocytes and neurons recapitulates features of TDP-43 proteinopathy C_LIO_LIGRN-/- cortical-like astrocytes are necessary and sufficient for the development of the TDP-43 loss of function phenotype in mbOrg. C_LIO_LIA TDP-43 phenotype can be rescued in neurons by treating neuron and astrocyte co-cultures with progranulin full length protein. C_LI eTOC blurbIn this article, de Majo and colleagues present a novel 3D iPSC-derived model to study neurodegenerative disorders such as ALS and FTD. When devoid of GRN expression, these cultures present features of ALS and FTD associated pathology hardly ever observed in vitro. These phenotypes are shown to be primarily driven by diseased astrocytes and can be rescued by progranulin supplementation.

neuroscience↗

bioRxiv2022

Genome Editing Is Induced in a Binary Manner in Single Human Cells

Even when precise nucleotide manipulations are intended, the outcomes of genome editing can be diverse, often including random insertions and deletions. The combinations and frequencies of these different outcomes in single cells are critical not only in the generation of genetically modified cell lines but also in the evaluation of the clinical effects of genome editing therapies. However, current methods only analyze cell populations, not single cells. Here, we utilized the Single Particle isolation System (SPiS) for the efficient isolation of single cells to systematically analyze genome editing results in individual human cultured cells. As a result, we discovered that genome editing induction has a binary nature, that is, the target alleles of cells tend to be all edited or not edited at all. This study enhances our understanding of the induction mechanism of genome editing and provides a new strategy to analyze genome editing outcomes in single cells.

bioengineering↗

bioRxiv2021

Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein aggregates within processing bodies

RNA binding motif protein 20 (RBM20) is a key regulator of alternative splicing in the heart, and its mutation leads to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic human induced pluripotent stem cells (iPSCs) with heterozygous or homozygous DCM-associated missense mutations in RBM20 (R636S) as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulated contractile dysfunction of RBM20-associated DCM and revealed greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP revealed a gain-of-function preference of mutant RBM20 for 3' UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing revealed that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO, impacting distinct cardiac signaling pathways. Splicing defects specific to KO or R636S mutations were supported by data from R636S gene-edited pig hearts and eCLIP. Super-resolution microscopy verified that mutant RBM20 maintains limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a novel pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways that are likely to mediate differential contractile phenotypes and stress-associated heart pathology.

cell biology↗

bioRxiv2020

Rapid, precise quantification of large DNA excisions and inversions by ddPCR

The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length. The method can be completed in a few hours without the need for next-generation sequencing. The ddXR method uncovered unexpectedly high rates of large (>20 kb) excisions and inversions, while also revealing a surprisingly low dependence on linear distance, up to 170 kb. We further modified the method to measure precise repair of excision junctions and allele-specific excision, with important implications for disease modeling and therapeutic gene editing.

molecular biology↗