Application of CRISPR/Cas9 system and the preferred no-indel end-joining repair in tardigrades

Kumagai, Hitomi; Kondo, Koyuki; Kunieda, Takekazu

doi:10.1016/j.bbrc.2022.07.060

Cited by 27 publications

(29 citation statements)

References 20 publications

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“…Recent progress in tardigrade transgenesis (S. Tanaka, Aoki, and Arakawa 2023) and promising findings of somatic mutagenesis by CRISPR-Cas9 (Kumagai, Kondo, and Kunieda 2022) are paving the way towards germ line gene editing in H. exemplaris . Knocking out Dsup and TDR1 genes should help to better appreciate their importance in radio-resistance and the underlying mechanisms.…”

Section: Discussionmentioning

confidence: 99%

Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation

Anoud,

Delagoutte,

Helleu

et al. 2023

Preprint

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Tardigrades, microscopic animals found in virtually all ecosystems, are renowned for their remarkable ability to withstand extreme conditions. Recent studies have identified novel tardigrade specific protein families that aid in resistance to desiccation and ionizing radiation (IR). Notably, a tardigrade specific DNA binding protein called Dsup (for DNA damage suppressor) has been found to protect from X-ray damage in human cells and from hydroxyl radicalsin vitro. However, Dsup has only been found in two species within the Hypsibioidea superfamily.To better understand mechanisms underlying radio-resistance in the Tardigrada phylum, we first characterized DNA damage and repair in response to IR in the model speciesHypsibius exemplaris. By analysis of phosphorylated H2AX, we demonstrated the induction and repair of DNA double-strand breaks after IR exposure. Importantly, the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in the remarkable radio-resistance of tardigrades. In order to identify novel tardigrade specific genes involved, we next conducted a comparative transcriptomics across three species,H. exemplaris,Acutuncus antarcticusandParamacrobiotus fairbanksi, the latter belonging to the Macrobiotoidea superfamily known to lack Dsup homologs. In all three species, many genes of DNA repair were among the most strongly overexpressed genes alongside a novel tardigrade specific gene, namedTardigradeDNA damageResponse protein1(TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and act by preserving chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade specific gene responsible for conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping to cope with high levels of DNA damage. Furthermore, it suggests that at least two tardigrade specific genes, respectively for Dsup and TDR1, have independently evolved DNA-binding functions that contribute to radio-resistance in the Tardigrada phylum.

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Section: Discussionmentioning

confidence: 99%

Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation

Anoud,

Delagoutte,

Helleu

et al. 2023

Preprint

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“…Work toward the latter has begun with the use of different live fluorescent dyes to temporarily label various cellular components, such as mitochondria, lysosomes, and membrane (McGreevy et al, 2018). Transgene and CRISPR techniques were recently developed for somatic cells of adult tardigrades, but they have not yet been demonstrated to work in germline cells (Kumagai et al, 2022; Tanaka et al, 2022). There has yet to be a technique established for labeling and live imaging of specific mRNA and/or proteins or for live reporting of mRNA transcription and/or translation in germline or embryos of this system.…”

Section: Discussionmentioning

confidence: 99%

The Embryonic Origin of Primordial Germ Cells in the TardigradeHypsibius exemplaris

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Smith

et al. 2023

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Primordial germ cells (PGCs) give rise to gametes – cells necessary for the propagation and fertility of diverse organisms. Current understanding of PGC development is limited to the small number of organisms whose PGCs have been identified and studied. Expanding the field to include little-studied taxa and emerging model organisms is important to understand the full breadth of the evolution of PGC development. In the phylum Tardigrada, no early cell lineages have been identified to date using molecular markers. This includes the PGC lineage. Here, we describe PGC development in the model tardigradeHypsibius exemplaris. The four earliest- internalizing cells (EICs) exhibit PGC-like behavior and nuclear morphology. The location of the EICs is enriched for mRNAs of conserved PGC markerswiwi1(water bearpiwi1) andvasa. At early stages, bothwiwi1andvasamRNAs are detectable uniformly in embryos, which suggests that these mRNAs do not serve as localized determinants for PGC specification. Only later arewiwi1andvasaenriched in the EICs. Finally, we traced the cells that give rise to the four PGCs. Our results reveal the embryonic origin of the PGCs ofH. exemplarisand provide the first molecular characterization of an early cell lineage in the tardigrade phylum. We anticipate that these observations will serve as a basis for characterizing the mechanisms of PGC development in this animal.

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“…The finding that storage cells undergo replication opens new possibilities for in vitro culture of this cell type and suggests its potential as a valuable model for studying cell cycle dynamics, responses to replication stress, and DNA damage control in tardigrades. With the advent of transgenesis (15) and CRISPR (46) technologies in tardigrades, along with the established RNAi methods (47, 48), Hypsibius exemplaris is increasingly becoming a promising genetic experimental model, altogether marking an exciting era in tardigrade research.…”

Section: Discussionmentioning

confidence: 99%

Storage cell proliferation during somatic growth establishes that tardigrades are not eutelic organisms

Quiroga-Artigas,

Moriel-Carretero

2023

Preprint

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Tardigrades, microscopic ecdysozoans renowned for their resilience to extreme environments, have long been thought to maintain a constant cell number after completing embryonic development, a phenomenon known as eutely. However, sporadic reports of dividing cells have raised questions about this assumption. In this study, we investigated whether tardigrades truly exhibit a fixed cell number during somatic growth using the model speciesHypsibius exemplaris. Comparing hatchlings to adults, we observed an overall increase in the number of storage cells, a tardigrade cell type involved in nutrient storage. To assess cell proliferation, we monitored DNA replication via the incorporation of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU). A significantly higher number of storage cells incorporated EdU while animals were still growing. Starvation halted both animal growth and storage cell proliferation, linking the two processes. Additionally, we found that EdU incorporation in storage cells is associated with molting, a critical process in tardigrade post-embryonic development, since it involves cuticle renewal to enable further growth. Finally, we show that hydroxyurea, a drug that slows down DNA replication progression, strongly reduces the number of EdU+cells and results in molting-related fatalities. Our data not only provide a comprehensive picture of replication events during tardigrade growth but also highlight the critical role of proper DNA replication in tardigrade molting and survival. This study definitively challenges the notion of eutely in tardigrades, offering promising avenues for exploring cell cycle, replication stress, and DNA damage management in these remarkable creatures as genetic manipulation techniques emerge within the tardigrade research field.

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Application of CRISPR/Cas9 system and the preferred no-indel end-joining repair in tardigrades

Cited by 27 publications

References 20 publications

Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation

Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation

The Embryonic Origin of Primordial Germ Cells in the TardigradeHypsibius exemplaris

Storage cell proliferation during somatic growth establishes that tardigrades are not eutelic organisms

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