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Efficiency of genome editing using modified single-stranded oligodeoxyribonucleotides in human cells

SCIENTIFIC REPORTS 2025年第1期15卷 1-9页

作者： Kim, Seryoung matsushita, yosuke Katagiri, Toyomasa Maseda, Hideaki Natl Inst Adv Ind Sci & Technol Biomed Res Inst 1-8-31 Midorigaoka Ikeda Osaka 5638577 Japan Tokushima Univ Inst Adv Med Sci Div Genome Med 3-18-18 Kuramotocho Tokushima 7708503 Japan Natl Inst Biomed Innovat Lab Biofunct Mol Med Hlth & Nutr 7-6-8 Saito Asagi Ibaraki Osaka 5670085 Japan Univ Teknol Malaysia Int Inst Technol Dept Environm Engn & Green Technol Kuala Lumpur 54100 Malaysia

Single-stranded oligodeoxyribonucleotide (ssODN) gene editing has emerged as a promising therapeutic strategy. However, further improvements in efficiency are desired for practical application. The effects of strand length and locked nucleic acid (LNA) modification on ssODN genome editing were investigated by introducing an assay cassette into the genome of HEK293T cells and measuring precise base deletions of eight bases. The introduction of LNAs into ssODNs, five pairs of LNAs at 25-35 nt from the centre and one pair at 20-25 nt, showed approximately 18-fold higher efficiency than unmodified ssODNs of the same length in the study using 70 nt ssODNs. In addition, genome editing efficiency was further improved when LNAs were introduced at the same positions as the 70 nt ssODN, which showed the highest efficiency for the 90 nt ssODN. However, in some cases, the same number of LNA modifications could conversely reduce the efficiency, and the modification positions in the ssODN method were successfully optimised in the present study. Furthermore, the oligo DNA was shown to be effective not only for deletions but also for base substitutions, with an editing efficiency of 0.63% per cell.

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Development and evaluation of a SYBR Green-based RT-qPCR assay with a specific primer set for tomato seed testing against tomato brown rugose fruit virus

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JOURNAL OF GENERAL PLANT PATHOLOGY 2025年第3期91卷 160-170页

作者： Ota, Emi Shinosaka, Hibiki Ishibashi, Kazuhiro Takeyama, Sawana Matsuyama, Momoko Tomitaka, Yasuhiro matsushita, yosuke Osaki, Kohei Kubota, Kenji NARO Inst Plant Protect Div Core Technol Pest Control Res 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan NARO Ctr Seeds & Seedlings Dept DUS Test & Seed Inspect 2-2 Fujimoto Tsukuba Ibaraki 3050852 Japan NARO Inst Agrobiol Sci Div Plant Mol Regulat Res 2-1-2 Kannondai Tsukuba Ibaraki 3058602 Japan NARO Inst Plant Protect Div Crop Pest Control Res 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan

Since its first occurrence in Israel and Jordan in 2014 and 2015, the tomato brown rugose fruit virus (ToBRFV) has become one of the most concerning pathogens affecting tomatoes and other crops worldwide. Its rapid spread is believed to result from the international trade of contaminated seeds and its seed transmissibility, underscoring the critical importance of seed health testing for ToBRFV to prevent further dissemination of the virus. To this end, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocols employing TaqMan probe chemistry have been widely adopted. However, the development of RT-qPCR protocols for ToBRFV seed testing using SYBR Green chemistry remains limited. The SYBR Green method offers the advantage of distinguishing ToBRFV from other tobamoviruses through melt curve analysis. In this study, we developed a SYBR Green-based RT-qPCR detection method using newly designed primer sets, which demonstrated high specificity for ToBRFV and sufficient sensitivity. While this protocol requires further optimization and validation for application in routine seed testing, it establishes a foundational approach for SYBR Green-based RT-qPCR seed testing. Additionally, this study raises an important question regarding the relationship between RT-qPCR results in seed tests and the likelihood of virus contamination or transmission via seeds.

关键词： ToBRFV Tobamoviruses SYBR green RT-qPCR Melt curve Tomato

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Characteristics of viroids infecting horticultural plants

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JOURNAL OF GENERAL PLANT PATHOLOGY 2016年第6期82卷 342-343页

作者： matsushita, yosuke NARO Inst Vegetable & Floriculture Sci Tsukuba Ibaraki 3050852 Japan

Viroids are the smallest and simplest plant pathogens, consisting of a single-stranded, circular, naked RNA genome, 246401 nucleotides long, lacking any protein-coding sequences (Ding 2009). The occurrence of pospiviroid diseases in Japan has previously been limited to those caused by Citrus exocortis viroid and Chrysanthemum stunt viroid. However, in July 2006, we found TCDVd-infected tomato plants in a farmers greenhouse in Hiroshima Prefecture, Japan (matsushita et al. 2008).

关键词： VIROIDS HORTICULTURAL crops PATHOGENIC microorganisms

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Host ranges and seed transmission of Tomato planta macho viroid and Pepper chat fruit viroid

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EUROPEAN JOURNAL OF PLANT PATHOLOGY 2017年第1期149卷 211-217页

作者： Yanagisawa, Hironobu matsushita, yosuke Natl Agr & Food Res Org Cent Reg Agr Res Ctr Tsukuba Ibaraki 3058666 Japan Iwate Univ United Grad Sch Agr Sci Morioka Iwate 0208550 Japan Natl Agr & Food Res Org Inst Vegetable & Floriculture Sci Tsukuba Ibaraki 3058519 Japan

Host ranges of Tomato planta macho viroid (TPMVd) and Pepper chat fruit viroid (PCFVd) were investigated across 32 species representing 10 genera, including horticultural plants that are frequently imported in large numbers by Japan. Plants were infected by mechanical sap inoculation, and viroid presence was checked using reverse-transcription polymerase chain reaction and back-inoculation of tomato. Among the 32 species, 16 and 15 were susceptible to TPMVd and PCFVd, respectively;most belonged to Solanaceae, and symptoms were only observed in tomato, potato, green pepper, and Solanum muricatum. Seed transmission of TPMVd and PCFVd was carried out using seeds obtained from infected tomato, Capsicum annuum, and Petunia x hybrida plants, and from a series of crosses involving healthy or infected Petunia x hybrida parental material. The seed transmission rate of TPMVd was 0-4.4% in tomato and 17.5 or 43.3% in Petunia x hybrida, while that of PCFVd was 0-1.4% in tomato and 0 or 16.8% in Petunia x hybrida. Seed transmission via the viroid-infected pollen parent or infected seed parent was investigated in Petunia x hybrida;rates were 91.8% and 100% for TPMVd and 69.2% and 65.3% for PCFVd, respectively. These data indicated a risk of unexpected worldwide viroid spread via international trade of asymptomatic plants and viroid-infected seeds and pollen.

关键词： PCFVd Petunia Seed transmission Solanaceae Tomato TPMVd

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Distribution of Tomato planta macho viroid in germinating pollen and transmitting tract

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VIRUS GENES 2018年第1期54卷 124-129页

作者： matsushita, yosuke Yanagisawa, Hironobu Natl Agr & Food Res Org Inst Vegetable & Floriculture Sci Tsukuba Ibaraki 3058519 Japan Natl Agr & Food Res Org Cent Reg Agr Res Ctr Tsukuba Ibaraki 3058666 Japan Iwate Univ United Grad Sch Agr Sci Morioka Iwate 0208550 Japan

Vertical and horizontal pollen transmission is important for efficient infection by viroids. Vertical pollen transmission of viroids is attributed to the infection by viroid in the embryo sac through infected pollen. To identify the viroid infection in pollen and pollen tubes elongating through the transmitting tract, we used in situ hybridization to histochemically analyze the distribution of Tomato planta macho viroid (TPMVd) in pollen grains, the stigma, and style of petunia plants. TPMVd was present in the generative nucleus and vegetative nucleus of mature infected pollen grains and germinating pollen grains. During pollen tube growth, TPMVd was present in the vegetative nucleus and two sperm nuclei, which were generated by division of the generative nucleus in the style transmitting tract. These findings indicated that viroid infection in sperm nuclei is responsible for vertical pollen transmission of viroids. TPMVd infection from TPMVd-infected pollen tubes to the transmitting tract was not observed. In addition, TPMVd signals were not confirmed in the stigma and transmitting tract of TPMVd-infected petunia plants, suggesting that viroids may not replicate in these tissues at the stage of mature style. Therefore, TPMVd may leak from the pollen tube somewhere in the ovary, except in the transmitting tract, during the horizontal transmission of TPMVd.

关键词： Horizontal transmission Petunia hybrida Sperm nuclei Transmitting tract Vertical transmission

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Effect of low temperature on the distribution of Chrysanthemum stunt viroid in Chrysanthemum morifolium

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PHYTOPARASITICA 2015年第5期43卷 609-614页

作者： matsushita, yosuke Shima, Yoshiteru NARO Inst Floricultural Sci Tsukuba Ibaraki 3058519 Japan Toyama Prefectural Agr Forestry & Fisheries Res C Toyama 9391327 Japan

Replication and cellular movement of viroids are dependent on host plant components. Low temperatures reduce the physiological activity of host plants and thus suppress the levels of viroids. Chrysanthemum stunt viroid (CSVd) concentration was reduced in infected chrysanthemum (Chrysanthemum morifolium) plants subjected to low-temperature treatment compared with untreated plants. In this study, we investigated the distribution patterns of CSVd in the developing shoot tips of CSVd-infected chrysanthemum plants subjected to one-month, three-month, and six-month low-temperature treatment using in situ hybridization. After one month of low-temperature treatment, strong CSVd signals were detected in leaf primordia (LP) and the apical dome (AD);after three months, weak CSVd signals were detected in LP and the AD;while after six months, CSVd signals were not detected in LP or the AD, but only in tissues within the vascular bundle directly beneath viroid-free LP and AD areas. These changes in the distribution of CSVd in shoot tips subjected to low-temperature treatment might result from a reduction in viroid replication and/or the inhibition of viroid intra- and intercellular movement.

关键词： Apical dome Callose deposition Chrysanthemum stunt viroid In situ hybridization Leaf primordia Shoot apical meristem

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Screening of Chrysanthemum seticuspe accessions reveals different degrees of resistance to chrysanthemum stunt viroid (vol 154, pg 1059, 2019)

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EUROPEAN JOURNAL OF PLANT PATHOLOGY 2019年第4期154卷 1201-1201页

作者： matsushita, yosuke Osaka, Masaaki Natl Agr & Food Res Org Inst Vegetable & Floriculture Sci Tsukuba Ibaraki 3058519 Japan Miyagi Prefectural Inst Agr & Hort Natori Miyagi 9811243 Japan

The chrysanthemum stunt viroid (CSVd) causes severe stunting and significant reduction in flower yield and quality of commercial chrysanthemum (Chrysanthemum morifolium). The pattern of inheritance and the detailed mechanism underlying CSVd resistance remains unclear. Chrysanthemum morifolium shows unstable and variable chromosome numbers that form a hexaploid complex with aneuploidy. To overcome the issue of complex hybridity and polyploidy, a diploid wild chrysanthemum, C. seticuspe f. boreale was used here as an alternative model to C. morifolium. We screened 20 C. seticuspe accessions for resistance to CSVd and identified one resistant accession “AET-19” showing resistance. Tissue print hybridization analysis showed that CSVd concentration decreased in the uppermost leaves of the AET-19 after the scions were cut from the infected rootstocks, suggesting the resistance seems to be ‘CSVd-disappearance type’. In situ hybridization experiments revealed that CSVd does not invade the shoot tip of AET-19 plants after graft-inoculation onto infected rootstocks. Taken together, the results highlight the usefulness of C. setiscupe as a model for analyzing the mechanism of CSVd resistance and its pattern of inheritance.

关键词： Chrysanthemum seticuspe CSVd Diploid Resistant Susceptible

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Seed transmission of potato spindle tuber viroid and its distribution in reproductive organs in Solanaceae weed species

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EUROPEAN JOURNAL OF PLANT PATHOLOGY 2023年第3期167卷 315-322页

作者： matsushita, yosuke Kubota, Kenji Natl Agr & Food Res Org NARO Inst Plant Protect Tsukuba Ibaraki 3058519 Japan

Potato spindle tuber viroid (PSTVd) is an important pathogen that severely affects tomato and potato production. Several Solanaceae species are naturally and experimentally susceptible to PSTVd, implying that Solanaceae weeds can be a reservoir of PSTVd through infected seeds in crop fields. To determine whether PSTVd is seed-transmissible in solanaceous weed species, we examined seed transmission rates and localization of PSTVd in the reproductive organs of Solanum nigrum and Physalis angulata. The results revealed that the seed transmission rate of PSTVd was 0%-50% and 89.7%-100% in S. nigrum and P. angulata, respectively, suggesting that these weed species act as reservoirs of PSTVd, and their seed seems to be an important source of PSTVd. While the seed transmission rate in S. nigrum was 0%-8% for PSTVd isolate VP35, it was 10.5%-50% for isolate VP72-1. In situ hybridization analysis showed that isolate VP72-1 (but not VP35) successfully invaded the ovule and embryo of S. nigrum, suggesting that the transmission rate in S. nigrum might depend on the distribution pattern of PSTVd in ovules and embryos. This is the first report to show the distribution patterns of a viroid in the reproductive organs of solanaceous weed species during their developmental stages.

关键词： Embryo Ovule Physalis angulata Physalis peruviana PSTVd Solanum nigrum

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TCP congestion control with ACK-pacing for vertical handover

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IEICE TRANSACTIONS ON COMMUNICATIONS 2007年第4期E90B卷 885-893页

作者： matsushita, yosuke Matsuda, Takahiro Yamamoto, Miki Osaka Univ Grad Sch Engn Suita Osaka 5650871 Japan Kansai Univ Fac Engn Suita Osaka 5648680 Japan

In this paper, we discuss TCP performance in a wireless overlay network where wireless LANs and cellular networks are integrated. In the overlay network, vertical handover, where a mobile node changes its access link during a session, is one of the most important technologies. When a vertical handover occurs, throughput performance of a TCP flow is degraded due to not only packet losses during the handover, but drastic change of its bandwidth-delay product. In this paper, we propose an ACK-pacing mechanism for TCP congestion control to improve the performance degradation. The proposed system is receiver-driven, so no modification is required to the mechanism of TCP sender. In the proposed system, a TCP receiver adjusts a transmission rate of ACKs according to the relationship between bandwidth-delay products before and after a handover. Since the ACK-clocking mechanism of TCP adjusts the transmission rate of TCP segments, the TCP receiver can seamlessly adjust its congestion window size to the new bandwidth-delay product. Computer simulation results show that the proposed system can improve the TCP performance during the vertical handover.

关键词： vertical handover TCP congestion control ACK-pacing

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Differences in dynamics of horizontal transmission of Tomato planta macho viroid and Potato spindle tuber viroid after pollination with viroid-infected pollen

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VIROLOGY 2018年 516卷 258-264页

For viroids, pollen transmission is an important transmission pathway to progeny seeds and new hosts. In the current study, we found that Tomato planta macho viroid (TPMVd)-but not Potato spindle tuber viroid (PSTVd)-was horizontally transmitted by pollen from petunia plants. Using tissue-printing hybridization to track the changes in viroid distribution after pollination, we noted that TPMVd was present in petunia stigma, styles, and eventually ovaries, whereas PSTVd was detected in the stigma and upper style but not the ovary. These findings suggest that horizontal transmission of viroids depends on the infection of the lower style and ovary during the elongation of pollen tubes after pollination. Additionally, TPMVd was transmitted horizontally, leading to systematic infection, when we used TPMVd-infected petunia pollen to pollinate the flowers of healthy tomato plants. Fertilization typically does not occur after heterologous pollination and thus likely is not required to accomplish horizontal transmission of viroids.

关键词： TPMVd PSTVd Pollen tube Horizontal transmission Vertical transmission Petunia hybrid Tomato Style Stigma Ovary

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