Biology subjects

Shirai, Y.

Publications and source records attributed to Shirai, Y..

3 recordsLinked to original sources

bioRxiv2026

A nuclear targeting approach enables efficient transgenesis in the milkweed bug Oncopeltus fasciatus

Insects show extreme diversity and have long intrigued biologists. Recent technological advancements, such as gene editing and transgenesis, should in principle enable the use of almost any insect species for biological research. However, in practice, species-specific challenges remain and conditions must be optimized carefully. Here, using the milkweed bug Oncopeltus fasciatus, we first generate a useful eye- and body-color mutant strain by using CRISPR/Cas9-mediated genome editing. Then we use this strain to develop an efficient piggyBac-mediated transgenesis system using a nuclear targeting approach. We show that incorporating 1x and 3x nuclear localization signals (NLS) into piggyBac mRNA substantially enhances overall transformation efficiency in O. fasciatus. Taking advantage of both the useful mutant strain and the efficient transgenesis system, we successfully integrated multiple expression cassettes ranging from 1.8 to 7.2 kb, including attP strains for phiC31-mediated site-specific integration and histone-labelled strains for live fluorescence imaging. We further provide evidence that the Q system, a binary expression system, is functional in this species, paving the way for future sophisticated genetic manipulations including functional assays for cis-regulatory elements. Together, our results not only expand the genetic toolkit of O. fasciatus as a comparative model insect, but also provide a practical framework for developing efficient transgenesis in other non-traditional model organisms.

developmental biology↗

bioRxiv2023

Quantification of the escape from X chromosome inactivation with the million cell-scale human single-cell omics datasets reveals heterogeneity of escape across cell types and tissues

One of the two X chromosomes of females is silenced through X chromosome inactivation (XCI) to compensate for the difference in the dosage between sexes. Among the X-linked genes, several genes escape from XCI, which could contribute to the differential gene expression between the sexes. However, the differences in the escape across cell types and tissues are still poorly characterized because no methods could directly evaluate the escape under a physiological condition at the cell-cluster resolution with versatile technology. Here, we developed a method, single-cell Level inactivated X chromosome mapping (scLinaX), which directly quantifies relative gene expression from the inactivated X chromosome with droplet-based single-cell RNA-sequencing (scRNA-seq) data. The scLinaX and differentially expressed genes analyses with the scRNA-seq datasets of [~]1,000,000 blood cells consistently identified the relatively strong degree of escape in lymphocytes compared to myeloid cells. An extension of scLinaX for multi-modal datasets, scLinaX-multi, suggested a stronger degree of escape in lymphocytes than myeloid cells at the chromatin-accessibility level with a 10X multiome dataset. The scLinaX analysis with the human multiple-organ scRNA-seq datasets also identified the relatively strong degree of escape from XCI in lymphoid tissues and lymphocytes. Finally, effect size comparisons of genome-wide association studies between sexes identified the larger effect sizes of the PRKX gene locus-lymphocyte counts association in females than males. This could suggest evidence of the underlying impact of escape on the genotype-phenotype association in humans. Overall, scLinaX and the quantified catalog of escape identified the heterogeneity of escape across cell types and tissues and would contribute to expanding the current understanding of the XCI, escape, and sex differences in gene regulation.

genomics↗

bioRxiv2023

DIPA-CRISPR gene editing in the yellow fever mosquito Aedes aegypti (Diptera: Culicidae)

Current methods for gene editing in insects rely on embryonic microinjection, which can be challenging for non-specialist laboratories. Recently, an alternative method known as "direct parental" CRISPR (DIPA-CRISPR) was developed. This method involves injecting commercial Cas9 protein and single-guide RNA into adult females, which can efficiently introduce mutations into developing oocytes. However, its versatility has not been fully explored, particularly in insects that have the most derived, polytrophic meroistic ovaries. In this study, we successfully applied DIPA-CRISPR to the yellow fever mosquito Aedes aegypti, which has polytrophic meroistic ovaries. Following adult injection of Cas9 ribonucleoproteins (Cas9 RNPs) targeting the kynurenine 3-monooxygenase gene, we recovered gene-edited G0 individuals. Injection at 24 h after blood-feeding resulted in the highest gene editing efficiency (3.5%), confirming that a key parameter of DIPA-CRISPR is the stage in which the adult females are injected. Together with our previous study, we demonstrated that DIPA-CRISPR is applicable to all three types of insect ovaries (i.e., panoistic, telotrophic, and polytrophic), which indicates that DIPA-CRISPR is a generalizable approach for insect gene editing.

genetics↗