Biology subjects

Ishiguro, S.

Publications and source records attributed to Ishiguro, S..

7 recordsLinked to original sources

bioRxiv2024

Repeatome landscapes and cytogenetics of hortensias provide a framework to trace Hydrangea evolution and domestication

Background and AimsOrnamental hortensias are bred from a reservoir of over 200 species in the genus Hydrangea s.l. and are valued in gardens, households and landscapes across the globe. The phenotypic diversity of hortensia cultivars, hybrids and wild relatives is mirrored by their genomic variation, with differences in genome size, base chromosome numbers and ploidy level. We aim to understand the genomic and chromosomal basis of hortensia genome variation. Therefore, we analyze six hortensias with different origins and chromosomal setups for repeatome divergence, the genome fraction with the highest sequence turnover. This holds information from the hortensias evolutionary paths and can inform breeding initiatives. MethodsWe compiled a hortensia genotype panel representing members of the sections Macrophyllae, Hydrangea, Asperae, and Heteromallae and reconstructed a plastome-based phylogenetic hypothesis as evolutionary basis for all our analyses. We comprehensively characterized the repeatomes by whole genome sequencing and comparative repeat clustering. Major tandem repeats were localized by multi-color FISH. Key ResultsThe Hydrangea species show differing repeat profiles reflecting their separation into the two major Hydrangea clades: Diploid Hydrangea species from Japan show a conserved repeat profile, distinguishing them from Japanese polyploids as well as Chinese and American hortensias. These results are in line with plastome-based phylogenies. The presence of specific repeats indicates that H. paniculata was not polyploidized directly from the common ancestor of Japanese Hydrangea species, but evolved from a distinct progenitor. Major satellite DNAs were detected over all H. macrophylla chromosomes. ConclusionsRepeat composition among the Hydrangea species varies in congruence with their origins and phylogeny. Identified species-specific satDNAs may be used as cytogenetic markers to identify Hydrangea species and cultivars, and to infer parental species of old Hydrangea varieties. This repeatome and cytogenetics information helps to expand the genetic toolbox for tracing hortensia evolution and informing future hortensia breeding.

genomics↗

bioRxiv2023

Pooled CRISPR screening of high-content cellular phenotypes by ghost cytometry

Fast enrichment of cells based on morphological information remains a challenge, limiting genome-wide perturbation screening for diverse high-content phenotypes of cells. Here we show that multi-modal ghost cytometry-based cell sorting is applicable to fast pooled CRISPR screening for both fluorescence and label-free high-content phenotypes of millions of cells. By employing the high-content cell sorter in the fluorescence mode, we enabled the genome-wide CRISPR screening of genes that affect NF-{kappa}B nuclear translocation. Furthermore, by employing the multi-parametric, label-free mode, we performed the large-scale screening to identify a gene involved in macrophage polarization. Especially the label-free platform can enrich target phenotypes without invasive staining, preserving untouched cells for downstream assays and unlocking the potential to screen for the cellular phenotypes even when suitable markers are lacking. One-Sentence SummaryMachine vision-based cell sorter enabled genome-wide perturbation screens for high-content cell phenotypes even without labeling

cell biology↗

bioRxiv2023

A multi-kingdom genetic barcoding system for precise target clone isolation

Clonal heterogeneity underlies diverse biological processes, including cancer progression, cell differentiation, and microbial evolution. Cell tagging strategies with DNA barcodes have recently enabled analysis of clone size dynamics and clone-restricted transcriptomic landscapes of heterogeneous populations. However, isolating a target clone that displays a specific phenotype from a complex population remains challenging. Here, we present a new multi-kingdom genetic barcoding system, CloneSelect, in which a target cell clone can be triggered to express a reporter gene for isolation through barcode-specific CRISPR base editing. In CloneSelect, cells are first barcoded and propagated so their subpopulation can be subjected to a given experiment. A clone that shows a phenotype or genotype of interest at a given time can then be isolated from the initial or subsequent cell pools stored throughout the experimental timecourse. This novel CRISPR-barcode genetics platform provides many new ways of analyzing and manipulating mammalian, yeast, and bacterial systems. TeaserA multi-kingdom CRISPR-activatable barcoding system enables the precise isolation of target barcode-labeled clones from a complex cell population.

synthetic biology↗

bioRxiv2022

The DNA-based global positioning system--a theoretical framework for large-scale spatial genomics

We present GPS-seq, a theoretical framework that enables massively scalable, optics-free spatial transcriptomics. GPS-seq combines data from high-throughput sequencing with manifold learning to obtain the spatial transcriptomic landscape of a given tissue section without optical microscopy. In this framework, similar to technologies like Slide-seq and 10X Visium, tissue samples are stamped on a surface of randomly-distributed DNA-barcoded spots (or beads). The transcriptomic sequences of proximal cells are fused to DNA barcodes, enabling the recovery of a transcriptomic pixel image by high-throughput sequencing. The barcode spots serve as "anchors" which also capture spatially diffused "satellite" barcodes, and therefore allow computational reconstruction of spot positions without optical sequencing or depositing barcodes to pre-specified positions. In theory, it could generate 100 mm x 100 mm spatial transcriptomic images with 10-20 m resolution by localizing 108 DNA-barcoded pixels with a single Illumina NovaSeq run. The general framework of GPS-seq is also compatible with standard single-cell (or single-nucleus) capture methods, and any modality of single-cell genomics, such as sci-ATAC-seq, could be transformed into spatial genomics in this strategy. We envision that GPS-seq will lead to breakthrough discoveries in diverse areas of biology by enabling organ-scale imaging of multiple genomic statuses at single-cell resolution for the first time.

bioinformatics↗

bioRxiv2019

A single CRISPR base editor to induce simultaneous C-to-T and A-to-G mutations

While several Cas9-derived base editors have been developed to induce either C-to-T or A-to-G mutation at target genomic sites, the possible genome editing space when using the current base editors remains limited. Here, we present a novel base editor, Target-ACE, which integrates the abilities of both of the previously developed C-to-T and A-to-G base editors by fusing an activation-induced cytidine deaminase (AID) and an engineered tRNA adenosine deaminase (TadA) to a catalytically impaired Streptococcus pyogenes Cas9. In mammalian cells, Target-ACE enabled heterologous editing of multiple bases in a small sequence window of target sites with increased efficiency compared with a mixture of two relevant base editor enzymes, each of which may block the same target DNA molecule from the other. Furthermore, by modeling editing patterns using deep sequencing data, the editing spectra of Target-ACE and other base editors were simulated across the human genome, demonstrating the highest potency of Target-ACE to edit amino acid coding patterns. Taking these findings together, Target-ACE is a new tool that broadens the capabilities for base editing for various applications.

bioengineering↗

bioRxiv2018

Fast and global detection of periodic sequence repeats in large genomic resources

Periodically repeating DNA and protein elements are involved in various important biological events including genomic evolution, gene regulation, protein complex formation, and immunity. Notably, the currently used genome editing tools such as ZFNs, TALENs, and CRISPRs are also all associated with periodically repeating biomolecules of natural organisms. Despite the biological importance of periodically repeating sequences and the expectation that new genome editing modules could be discovered from such periodical repeats, no software that globally detects such structured elements in large genomic resources in a high-throughput and unsupervised manner has been developed. Here, we developed new software, SPADE (Search for Patterned DNA Elements), that exhaustively explores periodic DNA and protein repeats from large-scale genomic datasets based on k-mer periodicity evaluation. SPADE precisely captured reported genome-editing-associated sequences and other protein families involving repeating domains with significantly better performance than the other software designed for limited sets of repetitive biomolecular sequences.

bioinformatics↗

bioRxiv2018

Genetic basis of transgressive segregation in rice heading phenotypes

Transgressive segregation produces hybrid progeny phenotypes that exceed parental phenotypes. Unlike heterosis, extreme phenotypes caused by transgressive segregation are heritably stable. We examined transgressive phenotypes of flowering time in rice. Our previous study examined days to flowering (heading; DTH) in six F2 populations for which the parents had distal DTH, and found very few transgressive phenotypes. Here, we demonstrate that transgressive segregation in F2 populations occurred between parents with proximal DTH. DTH phenotypes of the A58 x Kitaake F2 progenies frequently exceeded those of both parents. Both A58 and Kitaake are japonica rice cultivars adapted to Hokkaido, Japan, which is a high-latitude region, and have short DTH. Among the four known loci required for short DTH, three loci had common alleles in A58 and Kitaake, and only the one locus had different alleles. This result indicates that there is a similar genetic basis for DTH between the two varieties. We identified five new quantitative trait loci (QTLs) associated with transgressive DTH phenotypes by genome-wide single nucleotide polymorphism (SNP) analysis. Each of these QTLs showed different degrees of additive effects on DTH, and two QTLs had epistatic effect on each other. These results demonstrated that genome-wide SNP analysis facilitated detection of genetic loci associated with the extreme phenotypes and revealed that the transgressive phenotypes were produced by exchanging complementary alleles of a few minor QTLs in the similar parental genotypes.

genetics↗